Funded under USAID Cooperative Agreement No. 663-A-00-00-0358-00.

Produced in collaboration with the Ethiopia Public Health Training Initiative, The Carter

Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education.

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©2004 by Solomon Adugna, Lakshmi Ahuja Mekonnen Alemu, Tsehayneh Kelemu, Henok Tekola, Belayhun Kibret, Solomon Genet

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Table of Contents
Table of Contents	i
Acknowledgement	v
Introduction	vi
Solomon Adugna and Lakshmi Ahuja
Unit One: Enzymes	1
Henok Tekola
Principles and classifications of Enzymes	5
Mechanism action of Enzymes	7
Enzyme Inhibition	13
Regulation of Enzyme activity	18
Enzymes in clinical diagnosis	19
Unit Two: Carbohydrate Metabolism	23
Belayhun Kibret
Chemistry of Carbohydrates	23
Functions of Carbohydrates	31
Digestion and absorption of Carbohydrates	33
Glycolysis	35
Glycogen Metabolism	42
Glycogen storage diseases	45
Pentose phosphate pathway	46
The Cori - cycle	48
Gluconeogenesis	49
Unit Three: Integrative Metabolism Bioenergetics	54
Tsehayneh Kelemu
Introduction	54
Structural basis of high energy phosphate	55
Formation and utilization of ATP	56
Catabolism of fuel molecules	56
Concept of Free energy	58
Oxidation-reduction reactions	58
Aerobic energy generation	61
Kreb’s cycle	63
Function and regulation of kreb’s cycle	70

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Electron transport system and oxidative Phosphorylation	71
Respiratory control	77
Uncouplers	77
Respiratory poisons	78
Unit Four: Lipid Metabolism	80
Solomon Genet
Classification of Lipids	80
Types of Lipids	80
Digestion and absorption of Lipids	86
Metabolism of Fatty acids and Triacyl Glycerols	88
β-Oxidation of Fatty acids	90
Metabolism of Ketone bodies	93
Biosynthesis of Fatty acids and triacyl glycerols	97
Cholesterol Metabolism	99
Atherosclerosis	101
Hypercholesterolemic drugs	101
Lipid storage diseases	102
Fatty Liver	102
Lipoproteins	102
Chemical compositions of Membranes	104
Unit Five: Amino acids and Proteins	105
Solomon Adugna
Classification of amino acids	106
Properties of amino acids	115
Peptides	119
Glutathione synthesis	121
Proteins	124
Classifications of proteins	124
Levels of organization of proteins	126
Denaturation of proteins	131
Hemoglobin and Myoglobin	135
Sickle cell Hemoglobin	136
Sickle cell disease, Sickle cell trait	136
Digestion and absorption of proteins	137
Amino acid catabolism	141
Nitrogen balance, excretion and the urea cycle	144

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Defects in the urea cycle	150
The Glucose - Alanine cycle	151
Inborn errors of amino acid metabolism	152
Amino acid derived nitrogenous compounds	154
Clinical problems	159
Unit Six: Vitamins and Coenzymes	161
Solomon Adugna
Water soluble vitamins	161
Chemistry, sources, function and deficiency of:
Thamine	161
Riboflavin	163
Niacin	164
Pyridoxine	165
Biotin	166
Cobalamin	167
Folic acid	169
Panthothenic acid	170
Ascorbic acid	171
Fat soluble vitamins	172
Chemistry, Sources, function, deficiency and Hypervitaminosis of:
Vitamin A	173
Vitamin D	177
Vitamin E	179
Vitamin K	181
Unit Seven: Miniral Metabolism	183
Lakshmi Ahuja
Mineral	183
Sodium and potassium	183
Calcium and Phosphate	184
Trace elements	185
Iron	185
Copper	187
Magnesium	188
Flourine	188
Iodine	189
Zinc	189

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Selenium	191
Unit Eight: Hormones	193
Lakshmi Ahuja
Definition and classification	194
Biosynthesis, storage, transport	196
Mechanism of action of steroid hormones	197
Mechanism of action of protein hormones	198
Receptors and diseases	202
Second messengers	203
Insulin synthesis, secretion and metabolic role	205
Diabetes mellitus	209
Symptoms and complications of DM	210
Glucagon	211
Clinical aspects	212
Thyroxin	213
Synthesis and Metabolism	213
Hypo and Hyper Thyroidism	214
Goiter	215
Catecholamines	216
Pheochromocytoma	217
Case Histories	217
Unit Nine: Molecular Genetics	220
Mekonnen Alemu
Structure of Nucleic acids	220
Types of Nucleic acids	226
Replication of DNA	230
DNA Damage and repair mechanism	231
RNA synthesis	233
Post transcriptional Modifications	236
Translation/Protein synthesis	240
Regulation of protein synthesis	242
The Genetic code	243
Post translational processes	244
Glossary	246
Reference	253

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ACKNOWLEDGMENT

The team of authors from the department of Biochemistry of various Universities would like to thank and express their deep rooted gratitude to the Carter Center (EPHTI) for its initiation and drive to prepare this teaching material. The Center has not only supported the team financially, but also stood behind it firmly throughout the entire period of this experience. Thus Carter Center has become the pioneer in the field of preparing teaching material and also in training a team of authors for future endeavors of the kind.

In addition, the task would have been impossible without the directing of the Federal Democratic Republic of Ethiopia, Ministry of Education.

It is also not out of place to thank the administration of Gondar University, Debub University and Jimma University for extending cooperation whenever it was needed.

The authors extend their appreciation to Ato Akililu Mulugeta, Manager, Carter Center, who has shepherded the team’s effort through manuscript to finished pages.

The authors are particularly grateful to Abdel-Aziz Fatouh Mohammed (Ph.D) Associate professor of Biochemistry, Medical Faculty, Addis Ababa University and Ato. Daniel Seifu, Lecturer of Medical Biochemistry, Medical Faculty, Addis Ababa University for their highly professional editing and most helpful comments about many aspects of the text.

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INTRODUCTION TO BIOCHEMISTRY

Medical biochemistry is an essential component of curriculum for all categories of health professionals. Contemporary Biochemistry plays a crucial role in the Medical field, be it metabolic pathways, storage diseases, mechanism action of varied biomolecules or inter and intra cellular communications.

A lecture note on Medical biochemistry integrates and summarizes the essentials of the core subject. Topics are carefully selected to cover the essential areas of the subject for graduate level of Health sciences. The chapters are organized around the following major themes:

1.Conformation of biomolecules, structure and their relationship to biological activity

2.synthesis and degradation of major metabolites

3.Production and storage of energy

4.Biocatalysts and their application

5.Intercellular communication by hormones

6.Molecular events in gene expression and regulation

Enzymes:

Body proteins perform a large number of functions. One such unique function is, they act as biological catalysts (Enzymes) .They are responsible for highly complex reactions. They direct the metabolic events and exhibit specificity toward substrates, regulate the entire metabolism. Thus, they play key role in the degradation and synthesis of nutrients, biomolecules etc. The most important diagnostic procedures involves assay of enzymes. They assist to know damaged tissues, the extent of tissue damage, helps to monitor the course of the disease and used as a therapeutic means of diagnosing a vast array of diseases.

Amino acids and Proteins

Living systems are made up of Proteins .They are the dehydration polymers of amino acids. Each amino acid residue is joined by a peptide linkage to form proteins. Proteins are the molecular instruments in which genetic information is expressed, Hormones, Antibodies, transporters, the lens protein, the architectural framework of our tissues and a myriad of substances having distinct biological activities are derived. The type, nature and number of amino acids impart characteristic properties to the proteins.

There are about 300 amino acids, but only 20 are coded by DNA of higher organisms. Acid base properties of amino acids are important to the individual physical and chemical nature of

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the protein. The structural organization of proteins could be primary, secondary, tertiary and quaternary. The three dimensional structure is the most biologically active one.

The unfolding and disorganization of the proteins results in denaturation, the process is mostly irreversible. Such a protein may lose its biological function. Many amino acid derived peptides are of biological importance and special products formed from them are of critical importance to the body.

Carbohydrates

They are biomolecules, found abundantly in living organisms. They contain more than one hydroxyl group (polyhydric) In addition to aldehyde or ketone group. Thus, they form in to polyhydroxy aldoses or polyhydroxy ketoses. Carbohydrates can be classified in to Monosaccharide, disaccharide, and polysaccharides. Mono is the smallest sugar unit, disaccharide is made up of two monosaccharides joined by glycosidic linkages .The linkage can be α or β. A polymer with more than 10 monosaccharide units is called polysaccharide.

Carbohydrates have a wide range of functions. They provide energy; act as storage molecules of energy. Serve as cell membrane components and mediate some forms of communication between cells.

Absence of a single enzyme like lactase causes discomfort and diarrhea. The failure of Galactose and fructose metabolism due to deficient enzymes leads to turbidity of lens proteins (Cataract). Blood glucose is controlled by different hormones and metabolic processes. People suffer from Diabetes if the insulin hormone is less or not functioning well, such people are prone to atherosclerosis, vascular diseases, and renal failure.

Integrative Metabolism and Bioenergetics

Oxygen is utilized for the conversion of glucose to pyruvate. The same metabolite also forms from amino acid and protein metabolism. Other precursors like Glycerol, propionate can give rise to pyruvate. The main breakdown product of pyruvate is acetyl CoA, which is the common intermediate in the energy metabolism of carbohydrates, lipid and amino

central metabolic pathway, the Citric acid cycle in the mitochondrial matrix. Here it is converted to CO2, H2O and reduced coenzymes (NADH, FADH2). These reduced nucleotides are the substrates for oxidative Phosphorylation in mitochondria, their oxidation provide the energy for the synthesis of ATP, the free energy currency of cells.

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Lipids

The bulk of the living matter is made up of Lipids, carbohydrates and proteins.

Lipids are water insoluble, but can be extracted with non-polar solvents like Benzene, methanol, or ether.

Some lipids act as storage molecules for example triglycerides stored in adipose tissue. Transport forms of lipids (Lipoproteins),are present in combination with proteins

Building blocks of lipids are fatty acids. Some lipids like cholesterol lack fatty acids but are potentially related to them. Lipids are constituents of cell membrane and act as hydrophobic barrier that permits the entry/exit of certain molecules. Lipids carry fat soluble vitamins and form special biomolecules. Lipid imbalance can lead to serious diseases like obesity and atherosclerosis. Break down of fatty acid produce energy, excessive breakdown cause ketosis, ketoacidosis, coma and death.

Cholesterol level in blood is controlled by several regulatory mechanisms. Such information is applied in the treatment of patients with high cholesterol levels.

Vitamins and Minerals

They are organic compounds required in small quantities for the functioning of the body. They are not synthesized in the body, needed to be provided in the diet. Vitamins do not generate energy. Generally they are responsible for the maintenance of health and prevention of chronic diseases. Grossly there are two groups’ Water soluble vitamins are Vit. B-complex and C. Fat soluble vitamins are Vit A, D, E, and K.

Minerals are elements present in human body .Elements like C,H,N are provided by the diet and water .Second group includes Ca, P, Mg, Na, K. Cl and Sulphur. These are required in large quantities (100mg or more/day). They are called Macro elements.

A third group includes trace elements, which are required in small amounts for example Fe, I, Zn, etc.

Fluorine deficiency associated with tooth decay, excess of it causes fluorosis. Sources and requirement are of physiological importance. The metabolic role and deficiency disorders are important for the students of health sciences.

Vitamins and trace elements are particularly important for patients with gastrointestinal disorders, who are fed on artificial diets or parenteral nutrition.

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Hormones

Hormones are chemical messengers secreted by endocrine glands and specific tissues. They reach distant organs and stimulate or inhibit the function. They play important role in carrying messages to the various organs. They form part of a signaling system.

Hormones are synthesized in one tissue, secreted in to blood, transported as mobile messengers .When they reach target tissue, they exhibit their actions. Defect in the secretion, function, metabolism can lead to various diseases.

Molecular Biology

Human beings are highly developed species. How a person looks, behaves, suffers from diseases and the like are dictated by genetic material called DNA. The information is inherited from parent to offspring. The same is replicated from parent DNA to daughter DNA.

Individual characters are translated in to proteins, under the direction of DNA. First RNA is synthesized from DNA (Transcription) which is translated in to Proteins. These proteins are responsible for various metabolic functions. Protein expression during development, adaptation, aging and other related processes of life are controlled by DNA.

Changes in the genetic material cause hereditary diseases.

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„The coenzyme may participate in forming an intermediate enzyme-substrate complex

Example: NAD, FAD, Coenzyme A

Metal ions in enzymes

Many enzymes require metal ions like ca2+, K+, Mg2+, Fe2+, Cu2+, Zn2+, Mn2+ and Co2+ for their activity.

Metal-activated enzymes-form only loose and easily dissociable complexes with the metal and can easily release the metal without denaturation. Metalloenzymes hold the metal tightly on the molecule and do not release it even during extensive purification.

Metal ions promote enzyme action by

a.Maintaining or producing the active structural conformation of the enzyme (e.g. glutamine synthase)

b.Promoting the formation of the enzyme-substrate complex (Example: Enolase and carboxypeptidase A.)

c.Acting as electron donors or acceptors (Example: Fe-S proteins and cytochromes)

d.Causing distortions in the substrate or the enzyme Example: phosphotransferases).

Properties of Enzyme

A. Active site

Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid chains that create a three-dimensional surface complementary to the substrate.

The active site binds the substrate, forming an enzyme-substrate (ES) complex. ES is converted to enzyme-product (EP); which subsequently dissociates to enzyme and product.

For the combination with substrate, each enzyme is said to possess one or more active sites where the substrate can be taken up.

The active site of the enzyme may contain free hydroxyl group of serine, phenolic (hydroxyl) group of tyrosine, SH-thiol (Sulfhydryl) group of cysteine or imindazolle group of histidine to interact with there is substrates.

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It is also possible that the active site (Catalytic site) is different from the binding site in which case they are situated closely together in the enzyme molecule.

B. Catalytic efficiency/ Enzyme turnover number

Most enzyme- catalyzed reactions are highly efficient proceeding from 103 to 108 times faster than uncatalyzed reactions. Typically each enzyme molecule is capable of transforming 100 to 1000 substrate molecule in to product each second.

Enzyme turn over number refers to the amount of substrate converted per unit time (carbonic anhydrase is the fastest enzyme).

C. Specificity

Enzymes are specific for their substrate. Specificity of enzymes are divided into:

a. Absolute specificity:- this means one enzyme catalyzes or acts on only one substrate. For example: Urease catalyzes hydrolysis of urea but not thiourea.

b.Stereo specificity- some enzymes are specific to only one isomer even if the compound is one type of molecule:

For example: glucose oxidase catalyzes the oxidation of β-D-glucose but not α-D- glucose, and arginase catalyzes the hydrolysis of L-arginine but not D-arginine. *Maltase catalyzes the hydrolysis of α- but not β –glycosides.

Bond Specificity

*Enzymes that are specific for a bond or linkage such as ester, peptide or glycosidic belong to this group

Examples:

1.Esterases- acts on ester bonds

2.Peptidases-acts on peptide bonds

3.Glycosidases- acts on glycosidic bonds.

D. Regulation

Enzyme activity can be regulated- that is, enzyme can be, activated or inhibited so that the rate of product formation responds to the needs of the cell.

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E. Zymogens (- inactive form of enzyme)

Some enzymes are produced in nature in an inactive form which can be activated when they are required. Such type of enzymes are called Zymogens (Proenzymes).

Many of the digestive enzymes and enzymes concerned with blood coagulation are in this group

Examples: Pepsinogen - This zymogen is from gastric juice.When required Pepsinogen converts to Pepsin

Trypsinogen - This zymogen is found in the pancreatic juice, and when it is required gets converted to trypsin.

*The activation is brought about by specific ions or by other enzymes that are proteolytic.

Pepsinogen + H+ Pepsin

Trypsinogen Enteropeptidase Trypsin

Zymogen forms of enzymes a protective mechanism to prevent auto digestion of tissue producing the digestive enzymes and to prevent intravascular coagulation of blood.

F. Isoenzymes (Isozymes)

These are enzymes having similar catalytic activity, act on the same substrate and produces the same product but originated at different site and exhibiting different physical and chemical characteristics such as electrophoretic mobilities, amino acid composition and immunological behavior.

Example: LDH (Lactate dehydrogenase) exists in five different forms each having four polypeptide chains. H= Heart and M=Muscle.

Type	Polypeptide chain
LDH-1	H H H H
LDH-2	H H H M
LDH-3	H H M M
LDH-4	H M M M
LDH-5	M M M M

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Example. CPK (Creatine phospho kinase) exists in three different forms each having two polypeptide chains. Characteristic sub units are B=Brain and M= Muscle.

Type	Polypeptide chain
CPK-1	BB
CPK-2	MB
CPK-3	M

Classification of Enzymes

Enzymes are classified on the basis of the reactions they catalyze- Each enzyme is assigned a four-digit classification number and a systematic name, which identifies the reaction catalyzed.

The international union of Biochemistry and Molecular Biology developed a system of nomenclature on which enzymes are divided in to six major classes, each with numerous sub groups. Enzymes are classified based on the reactions they catalyze.

Each enzyme is characterized by a code number comprising four digits separated by points. The four digits characterize class, sub-class, sub-sub-class, and serial number of a particular enzyme.

Class I. Oxidoreducatases-

Enzymes catalyzing oxidation reduction reactions. Example: Lactate-dehydrogenase

1- Lactic acid + NAD+ Pyruvic acid + NADH+H+

Class II. Transferases:

Enzymes catalyzing a transfer of a group other than hydrogen (methyl, acyl, amino or phosphate groups)

Example: Enzymes catalyzing transfer of phosphorus containing groups.

ATP: D-hexose-6 phosphotransferase (Hexokinase)

ATP+D-Hexose ADP+D-hexose-6-phosphate

Example: Acetyl-CoA: Choline D-acyltransferase (Choline Acyltransferase)

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Class III. Hydrolases: Enzymes catalyzing hydrolysis of ester, ether, peptido, glycosyl, acid-anhydride, C-C, C-halide, or P-N-bonds by utilizing water.

Example: Enzymes action on glycosyl compounds

β-D- Galactoside galactohydrolase (β -Galactosidase)

αβ -D- Galactoside+H2O alcohol +D-Galactose

Class IV. Lyases: Enzymes that catalyze removal of groups from substances by mechanisms other than hydrolysis, leaving double bonds.

Enzymes acting on C-C, C-O, C-N, C-S and C-halide bonds.

Example : Carbon-Oxygen lyases

Malate hydro-lyase (Fumarase)

COOH			COOH
		CHOH
					CH + H2O + H2O
		CH2	HC
		COOH		COOH
Malate			Fumarate

Class V. Isomerases:

Includes all enzymes catalyzing interconversion of optical, geometric, or positional

isomers.

Example: Enzymes catalyzing interconversion of aldose and ketoses

D - Glyceraldehyde-3- phosphate ketoisomerase (triosephosphate isomerase)

D - Glyceraldehyde-3phosphate

Dihydroxyacetone phosphate.

Class VI. Ligases or synthetases.

Enzymes catalyzing the linking together of 2 compounds coupled to the breaking of a pyrophosphate bond in ATP or similar trinucleotides: GTP, UTP etc. included are enzymes catalyzing reactions forming C-O, C-S, C-N, and C-C bonds,

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Example: Enzymes catalyzing formation of C-N-bonds

L- Glutamine: ammonia ligase (ADP) [Glutamine Synthetase]

ATP + L-Glutamate + NH4 = ADP + orthophosphate + L-Glutamine

Example: Enzymes catalyzing formation of C-C bonds

Acetyl-CoA: CO2 ligase (ADP) [acetyl-CoA carboxylase] ATP+ Acetyl-COA-CO2 Malonyl-CoA+ADP+pi.

MECHANISM OF ACTION OF ENZYMES

Emil Fischer’s model Lock and Key model 1890.

Lock: Key model of enzyme action implies that the active site of the enzyme is complementary in shape to that of its substrate, i.e. the shape of the enzyme molecule and the substrate molecule should fit each other like a lock and Key

In 1958, Daniel Koshland, postulated another model; which implies that the shapes & the active sites of enzymes are complementary to that of the substrate only after the substrate is bound.

Figure: Models of enzyme- substrate interactions

Mechanism of Enzyme Action (1913)

Michaels and Menten have proposed a hypothesis for enzyme action, which is most acceptable. According to their hypothesis, the enzyme molecule (E) first combines with a substrate molecule (S) to form an enzyme substrate (ES) complex which further

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dissociates to form product (P) and enzyme (E) back. Enzyme once dissociated from the complex is free to combine with another molecule of substrate and form product in a similar way.

ENZYMES ENHANCE THE RATE OF REACTION BY LOWERING FREE ENERGY OF ACTIVAION

A chemical reaction S P (where S is the substrate and P is the product or products) will take place when a certain number of S molecules at any given instant posses enough energy to attain an activated condition called the “transition state”, in which the probability of making or breaking a chemical bond to form the product is very high.

The transition state is the top of the energy barrier separating the reactants and products. The rate of a given reaction will vary directly as the number of reactant molecules in the transition state. The “energy of activation is the amount of energy required to bring all the molecules in 1 gram-mole of a substrate at a given temperate to the transition state

A rise in temperature, by increasing thermal motion and energy, causes an increase in the number of molecules on the transition state and thus accelerates a chemical reaction. Addition of an enzyme or any catalyst can also bring about such acceleration.

The enzyme combines transiently with the substrate to produce a transient state having c lower energy of activation than that of substrate alone. This results in acceleration of the reaction. Once the products are formed, the enzyme (or catalyst) is free or regenerated to combine with another molecule of the substrate and repeat the process.

Activation energy is defined as the energy required to convert all molecules in one mole of reacting substance from the ground state to the transition state.

Enzyme are said to reduce the magnitude of this activation energy.

*During the formation of an ES complex, the substrate attaches itself to the specific active sites on the enzyme molecule by Reversible interactions formed by Electrostatic bonds, Hydrogen bonds, Vanderwaals forces, Hydrophobic interactions.

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Factors Affecting Enzyme Activity

Physical and chemical factors are affecting the enzyme activity. These include

1.Temperature

2.pH

3.Substrate/enzyme concentration etc.

Temperature

Starting from low temperature as the temperature increases to certain degree the

activity of the enzyme increases because the temperature increase the total energy of the chemical system .

There is an optimal temperature at which the reaction is most rapid (maximum). Above this the reaction rate decreases sharply, mainly due to denaturation of the enzyme by heat.

The temperature at which an enzyme shows maximum activity is known as the optimum temperature for the enzyme. For most body enzymes the optimum temperature is around 370c, which is body temperature.

Enzyme activity

Optimum temperature

10 20 30 40 50 Temperature

Figure. Effect of temperature on enzymatic reaction

1. Effect of pH

The concentration of H+ affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in an ionized or unionized sate in order to interact.

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For example, Catalytic activity may require that an amino-group of the enzyme be in the protonated form (-NH3+) At alkaline pH this group is deprotonated and the rate of reaction therefore declines.

Extreme pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid chains.

The pH at which maximum enzyme activity is achieved is different for different enzymes, and after reflects the pH+] at which the enzyme functions in the body.

For example, pepsin, a digestive enzyme in the stomach, has maximum action at pH 2, where as other enzymes, designed to work at neutral pH, are denatured by such an acidic environment.

Enzyme activity

Optimum pH

6	8
PH

Figure. Effect of pH on enzymatic reaction

3.Concentration of substrate

At fixed enzyme concentration pH and temperature the activity of enzymes is influenced by increase in substrate concentration.

An increase in the substrate concentration increases the enzyme activity till a maximum is reached. Further increase in substrate concentration does not increase rate of reaction.

This condition shows that as concentration of substrate is increased, the substrate molecule combine with all available enzyme molecules at their active site till not more active sites are available (The active Sites become saturated). At this state the enzyme is obtained it maximum rate (V max).

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Figure. Effect of Concentration of substrate on enzyme activity

The characteristic shape of the substrate saturation curve for an enzyme can be expressed mathematically by the Michaelis Menten equation:

K1 K2

E +S

ES

E +P

Km = K3 +K2 / K1

K-1

V= V max[S] Km + [S]

Where: V= Velocity at a given concentration of substrate (initial reaction velocity) Vmax = Maximal velocity possible with excess of substrate

[S] = concentration of the substrate at velocity V

Km = michaelis-constant of the enzyme for particular substrate.

Relationship between [S] and Km

Km shows the relationship between the substrate concentration and the velocity of the enzyme catalyzed reaction.

Take the point in which 50% of the active site of the enzyme will be saturated by substrate, Assume that at ½ Vmax-50% of the active site of enzyme becomes saturated. Therefore:

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Vo = ½ Vmax, at 50% saturation

½Vmax = Vmax[S]

Km + [S]

2[S] = Km + [S]

Km= [S]

Figure: Relationship between [S] and Km

Characteristics of Km

Km- can defined as the concentration of the substrate at which a given enzyme yields one-half its max. Velocity (i.e Km is numerically equal to the substrate concentration of which the reaction velocity equal to ½ Vmax)

Km- is characteristic of n enzyme and a particular substrate, and reflects the affinity of the enzyme for that substrate.

Km- values varies from enzyme to enzyme and used to characterized different enzymes.

Km- values of an enzyme helps to understand the nature and speed of the enzyme catalysis.

Small Km - A numerically small (Low) km reflects a high affinity of the enzyme for substrate because a low conc of substrate is needed to half saturate the enzyme- that is reach a velocity of ½ Vmax.

High Km - A numerically large (high) Km reflects a low affinity of enzyme for substrate b/c a high conc of substrate is needed to half saturate the enzyme.

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High Km Value f an enzyme means the catalysis of that enzyme is slow compared to low Km.

Km does not vary with the concentration of enzyme.

4. Relationship of Velocity to Enzyme Concentration

The rate of the reaction is directly proportional to enzyme concentration at all substrate concentration. For example, if the enzyme concentration halved, the initial rate of the reaction (Vo) is reduced to one half that of the original.

Enzyme activity

Enzyme concentration

Figure. Effect of Enzyme concentration on enzymatic reaction

Order of Reaction

When [S] is much less than Km, the velocity of the reaction is roughly proportional to the substrate concentration. The rate of reaction is then said to be first order configuration with respect to substrate. When [S] is much greater than Km, the velocity is constant and equal to V max. The rate of reaction is then independent of substrate concentration and said to be zero order with respect to substrate concentration.

Enzyme Inhibition

Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor and the process is known as inhibition.

There are two major types of enzyme inhibition, Irreversible and Reversible.

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Irreversible Inhibition

The type of inhibition that can not be reversed by increasing substrate concentration or removing the remaining free inhibitor is called Irreversible inhibition

Eg. Diisopropyl & luorophosphate (DFP) Inhibits the enzyme acetyl cholinesterase, important in the transmission of nerve impulses. Acetyl cholinesterase catalyzes the hydrolysis of Acetylcholin (to acetic acid and choline) a neurotransmitter substance functioning in certain portions of the nervous system

„DEP inhibits also trypsin, chymotrypsin elastase, and phosphglucomutase

Organo-phosphorus compounds like malathion, parathron pesticides-inhibits acetyl cholinesterase by the same way as DFP.

Example: Inhibition of triose phosphate dehydrogenate by iodo acetate which block the activity of the enzyme.

REVERSIBLE INHIBITION

This type of inhibition can be Competitive, Non-competitive and uncompetitive Competitive Inhibition: This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy, therefore, competes with the substrate for that site.

In competitive inhibition the inhibitor and substrate compete for the same active site on the enzyme as a result of similarity in structure. The enzyme substrate complex will be broken dawn to products (E+S'ESE+P) where as enzyme inhibitor complex; (EI) will not be broken down to products.

A classical example is Malonate that competes with succinate and inhibits the action of succinate dehydrogenase to produce fumarate in the Krebs cycle.

The enzyme can be also inhibited by oxalate and glutarate because of the similarity of this substance with succinate

Eg.2 Allopurinol used for the treatment of Gout

Allopurinol Inhibits Xanthine oxidase by competing with Uric acid precursors for the active site on the enzyme. This competition blocks the conversion of these precursors, and of hypoxanthine and xanthine, to uric acid and result in lower serum urate levels.

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Inhibition of Enzyme Catalyzed Reactions

To avoid dealing with curvilinear plots of enzyme catalyzed reactions, biochemists Lineweaver and Burk introduced an analysis of enzyme kinetics based on the following rearrangement of the Michaelis-Menten equation:

[1/v] = [Km (1)/ Vmax[S] + (1)/Vmax]

Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an

intercept on the ordinate at 1/Vmax.

A Lineweaver-Burk Plot

An alternative linear transformation of the Michaelis-Menten equation is the

Eadie-Hofstee transformation:

v/[S] = -v [1/Km] + [Vmax/Km]

and when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a linear plot with a slope of -1/Km and the value Vmax/Km as the intercept on the y- axis and Vmax as the intercept on the x-axis.

Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menton equation are useful in the analysis of enzyme inhibition. Since most clinical drug therapy is based on inhibiting the activity of enzymes, analysis of enzyme reactions using the tools described above has been fundamental to the modern design of pharmaceuticals

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Effect of Competitive inhibitors

1.Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing [s]. at a sufficiently high substrate concentration, the reaction velocity reaches the Vmax. observed in the absence of inhibitor.

2.Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that in the presence of a competitive inhibitor more substrate is needed to achieve ½ Vmax.

Figure: Competitive inhibition

Non-Competitive Inhibition

In non-competitive inhibition the inhibitor binds at different site rather than the substrate-binding site. When the inhibitor binds at this site there will be a change in conformation of the enzyme molecules, which leads to the reversible inactivation of the catalytic site. Non-competitive inhibitors bind reversibly either to the free-enzyme or the ES complex to form the inactive complexes EI and ESI (Enzyme substrate Inhibition)

The most important non-competitive inhibitors are naturally occurring metabolic intermediates that can combine reversibly with specific sites on certain regulatory enzymes, that changes the activity of their catalytic sites.

An Example: is the inhibition of L-threonine dehydratase by L-isoleucine.

*Such type of Enzyme is called Allosteric Enzyme, which has a specific sites or allosteric site other than the substrate-binding site.

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1.Effect on Vmax.

Non-Competitive inhibition cannot be overcome by increasing the concentration of substrate. Thus, non-competitive inhibitors decrease the Vmas of the reaction.

2.Effect on Km:

Non-competitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the non- competitive inhibitor.

Figure: Noncompetitive inhibition

Uncompetitive Inhibition

Uncompetitive Inhibitor binds only to ES complex at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor-binding site available. Inhibition cannot be reversed by substrate.

In this case apparent Vmax. and Km decreased.

Figure: Uncompetitive inhibition

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Regulation of enzyme activity

There are several means by which the activity of a particular enzyme is specifically regulated.

1. Irreversible covalent Activation / Zymogen activation

Some enzymes are secreted in an inactive form called Proenzymes or zymogens. At

the site of action specific peptide bonds are hydrolysed either enzymatically or by PH

changes to convert it into active form, e.g. Pepsinogen Æ pepsin, TrypsinogenÆ

trypsin, plasminogenÆ plasmin. After hydrolysis when it is activated, it cannot be

reconverted into proenzyme form.

2. Reversible Covalent Modification

By addition of or removal of phosphate or adenylate, certain enzymes are reversibly

activated and inactivated as per the requirement. Protein kinase of muscle

phosphorylate phosphorylase kinase, glycogen synthetase by making use of ATP.

3.Allosteric Modulation

In addition to simple enzymes that interact only with substrates and inhibitors, there is a class of enzymes that bind small, physiologically important molecules and modulate activity in ways other than those described above. These are known as allosteric enzymes; the small regulatory molecules to which they bind are known as effectors. Allosteric effectors bring about catalytic modification by binding to the enzyme at distinct allosteric sites, well removed from the catalytic site, and causing conformational changes that are transmitted through the bulk of the protein to the catalytically active site(s).

The hallmark of effectors is that when they bind to enzymes, they alter the catalytic properties of an enzyme's active site. Those that increase catalytic activity are known as positive effectors. Effectors that reduce or inhibit catalytic activity are negative effectors.

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There are two ways that enzymatic activity can be altered by effectors: the Vmax can be increased or decreased, or the Km can be raised or lowered.

4. Feedback inhibition

In allosteric regulation in which end products inhibit the activity of the enzyme is called” feedback inhibition”.

A high conc. D typically inhibits conversion of AÆ B.

This involves not simple backing up of intermediates but the activity of D to bind to and inhibit E1. D thus acts as negative allosteric affector or feedback inhibitor of E1.

The kinetics of feedback inhibition cay be competitive, mixed, etc. It is the commonest way of regulation of a biosynthetic pathway. Feedback regulation generally occurs at the earliest functionally irreversible step unique in the biosynthetic pathway.

ENZYMES IN CLINICAL DIAGNOSIS

Plasma enzymes can be classified into two major groups

1.Those, relatively, small group of enzymes secreted into the plasma by certain organs (i.e. Enzymes those have function in plasma) For example: - the liver secretes zymogens of the enzymes involved in blood coagulation.

2.Those large enzyme species released from cells during normal cell turnover. These enzymes are normally intracellular and have no physiologic function in the plasma. In healthy individuals the levels of these enzymes are fairly constant and represent steady state in which the rate of release from cells into the plasma is balanced by an equal rate or removal from the plasma.

Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes are routinely

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determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Thus, the degree of elevation of a particular enzyme activity in plasma is often useful in evaluating the diagnosis and prognosis for the patient.

Measurement of enzymes concentration of mostly the latter type in plasma gives valuable informatio0n about disease involving tissues of their origin.

1. Lipase:

It is an enzyme catalyzing the hydrolysis of fats. It is secreted by pancreas and Liver. The plasma lipase level may be low in liver disease, Vitamin A deficiency, some malignancies, and diabetes mellitus. It may be elevated in acute pancreatitis and pancreatic carcinoma.

2. α- Amylase

α- amylase is the enzyme concerned with the break down of dietary starch and glycogen to maltose. It is present in pancreatic juice and saliva as well as in liver fallopian tubes and muscles. The enzyme is excreted in the Urine. The main use of amylase estimations is in the diagnosis of acute pancreatitis. The plasma amylase level may be low in liver disease and increased in high intestinal obstruction, mumps, acute pancreatitis and diabetes.

3. Trypsin

Trypsin is secreted by pancreas. Elevated levels of trypsin in plasma occur during acute pancreatic disease.

4. Alkaline phosphates (ALP)

The alkaline phosphates are a group of enzymes, which hydrolyze phosphate esters at an alkaline pH. They are found in bone, liver, kidney, intestinal wall, lactating mammary gland and placenta. In bone the enzyme is found in osteoblasts and is probably

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important for normal bone function. The level of these enzymes may be increased in rickets and osteomalacia, hyperparathyroidism, paget's disease of bone, obstructive jaundice, and metastatic carcinoma. Serum alkaline phosphatase levels may be increase in congestive heart failure result of injury to the liver.

5. Acid Phosphatase (ACP)

Acid phosphatases catalyzing the hydrolysis of various phosphate esters at acidic pH is found in the prostate, liver, red cells, platelets and bone. It may be elevated in metastatic prostatic carcinoma.

6. Transaminases

Two transaminases are of clinical interest.

1.Aspartate Transaminase, AST ( Glutamate oxaloacetate transaminase, GOT ) catalyzes the transfer of the amino group of aspartic acid to α- ketoglutarate forming glutamate and oxaloacetate.

AST or GOT is widely distributed, with high concentration, in the heart, liver, skeletal muscle, kidney and erythrocytes, and damage to any of these tissues may cause raised levels.

2.Alanine transaminase, ALT (Glutamate pyruvate transaminase, GPT ) Transfer the amino group of alanine to α- ketoglutarate, forming glutamate and pyruvate. It is present in high concentration in liver and to a lesser extent in skeletal muscle, kidney and heart.

-Serum levels of glutamate- pyruvate transaminase (SGOT) and Glutamate- oxaloacetate- transaminase (SGOT) are useful in the diagnosis of liver parenchymal damage and myocardial damage respectively. In liver damage, both enzymes are increased, but SGPT increases more. In myocardial infarction SGOT is increased with little or no increase in SGPT.

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7. Lactate Dehydrogenase (LDH)

It catalyzes the reversible interconversion of lactate and pyruvate. It is widely distributed with high concentrations in the heart, skeletal muscle, liver, kidney, brain and erythrocytes.

The enzyme is increased in plasma in myocardial infarction, acute leukemias, generalized carcinomatosis and in acute hepatitis. Estimation of it isoenzymes is more useful in clinical diagnosis to differentiate hepatic disease and myocardial infarction.

8. Creatine kinase (CK) or ceratin phosphokinase (CPK)

CK (CPK) is found in heart muscle brain and skeletal muscle. Measurement of serum creatine phosphokinase activity is of value in the diagnosis of disorders affecting skeletal and cardiac muscle. The level of CPK in plasma highly increased in myocardial infarction.

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UNIT TWO

CARBOHYDRATS

Objectives

•Define carbohydrates in chemical terms

•Classify carbohydrates in to three major groups with examples of each group

•List the monosaccharides of biological importance and learn their properties

•List the disaccharides of biological importance and learn their properties

•List the polysaccharides of biological importance and learn their properties

•Study the chemistry and functions of glycoproteins

Introduction

Carbohydrates are the most abundant macromolecules in nature. They are the main source and storage of energy in the body. They serve also as structural component of cell membrane. The general molecular formula of carbohydrate is CnH2nOn or (CH2O)n, where n > 3. Chemically, they contain the elements Carbon, hydrogen and oxygen. Thus they are Carbon compounds that contain large quantities of Hydroxyl groups.

Carbohydrates in general are polyhydroxy aldehydes or ketones or compounds which give these substances on hydrolysis.

Chemistry of Carbohydrates

Classification and Structure

Classification

There are three major classes of carbohydrates

•Monosaccharides (Greek, mono = one)

•Oligosaccharides (Greek, oligo= few) 2-10 monosaccharide units.

•Polysaccharides (Greek, Poly = many) >10 monosaccharide units.

Monosaccharides

Monosaccharides also called simple sugars. They consist of a single polyhydroxy aldehyde or ketone units. The most abundant monosaccharides in nature are the 6-carbon sugars like D- glucose and fructose.

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Structure

Monosaccharide has a backbone, which is un- branched, single bonded carbon chain. One of the carbon atoms is double bonded to an oxygen atom to form carbonyl group. Each of the other carbon atoms has a hydroxyl group. Example. Structure of Glucose

Open chain D-glucose	α-D –glucose	α-D –glucose
	(Fisher formula)	(Haworth formula)
Fig.2.1. structures of D.Glucose

There are two families of monosaccharides. Monosaccharides having aldehyde groups are called Aldoses and monosaccharides with Ketone group are Ketoses.

Depending on the number of carbon atoms, the monosaccharides are named trioses (C3), tetroses (C4), pentoses (C5), hexoses (C6), heptoses (C7).

No of carbon atoms	Generic name	Aldose family	Ketose family
3	Triose	Aldotriose	Ketotriose
		Eg.Glyceraldehyde.	Eg. Dihydroxyacetone
4	Tetrose	Aldotetrose	Ketotetrose
		Eg. Erythrose	Eg. Eyrthrulose
5	Pentose	Aldopentose	Ketopentose
		Eg. Ribose	Eg. Ribulose,Xylulose
6	Hexose	Aldohexose	Ketohexose
		Eg. Glucose	Eg. Fructose
		Galactose
		Mannose

Table 1.1 Common Biologically important monosaccharides with their families.

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Physical properties

Physical properties of Monosaccharides

They are colorless, crystalline compounds, readily soluble in water. Their solutions are optically active and exhibit the phenomenon of mutarotation. Carbohydrates spontaneously change between the α and β configuration.

Asymmetric Center and Stereoisomerism

Asymmetric carbon is a carbon that has four different groups or atoms attached to it and having optically activity in solution.

All the monosaccharides except dihydroxyacetone contain one or more asymmetric or chiral carbon atoms and thus occur in optically active isomeric forms. Monosaccharides with n number of asymmetric centers will have (2n) isomeric forms. (n= number of asymmetric carbon atoms).

Fig 2.2: The two isomeric forms of glyceraldehyde.

The designation of a sugar isomer as the D- form or of its mirror images the L- form is determined by the spatial relationship to the parent compound of the carbohydrate family. The D and L forms of Glyceraldehyde are shown in the Figure 2.2.The orientation of- OH and- H groups around the carbon atom adjacent to the terminal primary alcohol carbon determines its D or L form .When the - OH group on this carbon is on the right, the sugar is a member of the D- series, when it is on the left, it is a member of the L-series. These D and L configuration are also called Enantiomers.

Optical Activity

The presence of asymmetric carbon atom causes optical activity. When a beam of plane- polarized light is passed through a solution of carbohydrate it will rotate the light either to right or to left. Depending on the rotation, molecules are called dextrorotatory (+) (d) or levorotatory (-)

(l). Thus, D- glucose is dextrorotatory but D- fructose is levorotatory. When equal amounts of D

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and L isomers are present, the resulting mixture has no optical activity, since the activities of each isomer cancel one another. Such a mixture is called racemic or DL mixture.

Epimers

When sugars are different from one another, only in configuration with regard to a single carbon atom (around one carbon atom) they are called epimers of each other. For example glucose and mannose are epimers. They differ only in configuration around C2. Mannose and Galactose are epimers of Glucose

Fig 2.3: Structure of D - glucose, D-mannose and D- Galactose.

Anomers

The two stereoisomers at the hemiacetal (anomeric) carbon are:

oThe alpha anomer: Where- OH group is down (Haworth) o The beta anomer:Where- OH group is up (Haworth)

•Anomers are diastereomers (having different physical properties)

Cyclization of monosaccharides

Monosaccharides with five or more carbon atoms in the backbone usually occur in solution as cyclic or ring structure, in which the carbonyl group is not free as written on the open chain structure but has formed a covalent bond with one of the hydroxyl group along the chain to form a hemiacetal or hemiketal ring. In general, an aldehyde can react with an alcohol to form a hemiacetal or acetal.

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The C-1 aldehyde in the open-chain form of glucose reacts with the -5th carbon atom containing hydroxyl group to form an intramolecular hemiacetal. The resulting six membered ring is called pyranose because of its similarity to organic molecule Pyran.

Two different forms of glucose are formed when the OH group extends to right it is α-D-Glucose and when it extends to left, it is β-D-Glucose commonly called as Anomers.

Similarly, a ketone can react with an alcohol to form a hemiketal or ketal.

The C-2 keto group in the open chain form of fructose can react with the 5th carbon atom containing hydroxyl group to form an intramolecular hemiketal. This five membered ring is called furanose because of its similarity to organic molecule furan

Fig 2.4. α and β forms of Fructose

Oligosaccharides

Oligosaccharides contain 2 to 10 monosaccharide units. The most abundant oligosaccharides found in nature are the Disaccharides.

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Disaccharides

When two monosaccharides are covalently bonded together by glycosidic linkages a disaccharide is formed. Glycosidic bond is formed when the hydroxyl group on one of the sugars reacts with the anomeric carbon on the second sugar.

Biologically important disaccharides are sucrose, maltose, and Lactose.

Maltose

Maltose contains two D glucose residues joined by a glycosidic linkage between OH at the first carbon atom of the first glucose residues and OH at the fourth carbon atom of the second glucose forming a α-(1,4) glycosidic linkage as shown in Figure below. Maltose is the major degradative product of Starch.Maltose is hydrolyzed to two molecules of D- glucose by the intestinal enzyme maltase, which is specific for the α- (1, 4) glycosidic bond.

Fig 2.5. Structure of Maltose

Lactose

Lactose is a disaccharide of β-D galactose and β-D- glucose which are linked by β-(1,4) glycosidic linkage. Lactose acts as a reducing substance since it has a free carbonyl group on the glucose. It is found exclusively in milk of mammals (Milk sugar).

Figure2.6: Structure of Lactose

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Sucrose (Cane sugar)

Sucrose is a disaccharide of α- D- glucose and β-D-fructose. It is obtained from cane sugar. It is also present in various fruits. In contrast to other disaccharides sucrose contains no free anomeric carbon atom. Since the anomeric carbons of both its component monosaccharide units are linked to each other. For this reason sucrose is non reducing sugar.

Fig 2.7. Structure of sucrose α-(1, 2) β-Glycosidic bond

Polysaccharides

Most of the carbohydrates found in nature occur in the form of high molecular polymers called polysaccharides.

There are two types of polysaccharides .These are:

•Homopolysaccharides that contain only one type of monosaccharide building blocks.

•Heteropolysaccharides, which contain two or more different kinds monosaccharide building blocks.

Homopolysaccharides

Example of Homopolysaccharides: Starch, glycogen, Cellulose and dextrins.

Starch

It is one of the most important storage polysaccharide in plant cells.It is especially abundant in tubers, such as potatoes and in seeds such as cereals.

Starch consists of two polymeric units made of glucose called Amylose and Amylopectin but they differ in molecular architecture.

Amylose is unbranched with 250 to 300 D-Glucose units linked by α-(1, 4) linkages Amylopectin consists of long branched glucose residue (units) with higher molecular weight.

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The inner part of glucose units in amylopectin are joined by α-(1,4) glycosidic linkage as in amylose , but the branch points of amylopectin are α- (1,6) linkages. The branch points repeat about every 20 to 30 (1-4) linkages

Glycogen

-Glycogen is the main storage polysaccharide of animal cells (Animal starch).

-It is present in liver and in skeletal muscle.

- Like amylopectin glycogen is a branched polysaccharide of D-glucose units in α - (1, 4) linkages, but it is highly branched.

-The branches are formed by α -(1,6) glycosidic linkage that occurs after every 8 -12 residues. Therefore liver cell can store glycogen within a small space. Multiple terminals of branch points release many glucose units in short time.

Cellulose

Cellulose is the most abundant structural polysaccharide in plants. It is fibrous, tough, water insoluble. Cellulose is a linear unbranched homopolysaccharide of 10,000 or more D- glucose units connected by β-(1, 4) glycosidic bonds. Humans cannot use cellulose because they lack of enzyme (cellulase) to hydrolyze the β-( 1-4) linkages.

Figure: Structure of Cellulose

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Dextrins

These are highly branched homopolymers of glucose units with α-(1, 6), α-(1, 4) and α-(1, 3) linkages. Since they do not easily go out of vascular compartment they are used for intravenous infusion as plasma volume expander in the treatment of hypovolumic shock.

Hetero polysaccharides

These are polysaccharides containing more than one type of sugar residues 1. Glycosaminoglycans, (GAGs or mucopolysaccharides)

They are long, usually unbranched, composed of a repeating disaccharide units

*They are negatively charged heteroplolysaccharid chains (polyanions)

•The amino sugar is either D-glucosamine or D-galactosamine in which the amino group is usually acetylated, thus eliminating its positive charges.

•The amino sugar may also be sulfated on carbon 4, 6, or on a monoacetylated nitrogen.

•The acidic sugar is either D-glucuronic acid or its carbon 6 epimer, L-uronic acid. For example Hayluronic acid, Heparin and chondatin sulphate.

Function of Glycosammoglycans. (GAGS)

1.They have the special ability to bind large amounts of water, there by producing the gel-like matrix that forms the basis of the body’s ground substance.

2.Since they are negatively charged, for example, in bone, glycosaminoglycans attract and tightly bind cattions like ca++, they also take-up Na+and K+

3.GAGs stabilize and support cellular and fibrous components of tissue while helping maintain the water and salt balance of the body.

4.Its essential components of the extra cellular matrix, GAGs’ play an important role in mediating cell-cell interactions

•Ground substance is a part of connective tissue, which is a gel like substance containing

water, salt, proteins and polysaccharides.

An example of specialized ground substance is the synovial fluid, which serves as a lubricant in joints, and tendon sheaths.

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3.Heparin:

•contains a repeating unit of D-glucuronic and D-gluconsamine, with sulfate groups on some of the hydroxyl and aminx-groups

•It is an important anticoagualtn, prevents the clotting of blood by inhiginting the conversion of prothrombin to throbin. Thrombin is an enzyme that acts on the conversion of plasma fibrinogen into the fibrin.

•It is found in mast cells in lung, liver skin and intestinal mucosa.

Glycoproteins (Mucoproteins)

Glycoprotiens are proteins to which oligosaccharides are covalently attached. They differ from the glycosaminoglycans in that the length of the glycoproteins carbohydrate chain is relatively short (usually two to ten sugar residues in length, although they can be longer), whereas it can be very long in the glycosaminoglycans.

The glycoprotein carbohydrate chains are often branched instead of linear and may or may not be negatively charged.

For example:

-Glycophorin, a glycoprotein found in human red cell membranes.

-Human gastric glycoprotein (mucin).

-Many protein hormones,receptors are glycoproteins

Proteoglycans

When glycosamnoglycans are attached to a protein molecule the compound is called proteoglycan [proteoglycans = Glycosaminoglycans + proteins]

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Absorption of Carbohydrates

Products of digestion of dietary carbohydrates are practically completely absorbed almost entirely from the small intestine.

Absorption from proximal jejunum is three times grater than that of distal ileum. It is also proved that some disaccharides, which escape digestion, may enter the cells of the intestinal lumen by “pinocytosis” and are hydrolyzed within these cells. No carbohydrates higher than the monosaccharides can be absorbed directly in to the blood stream.

Mechanism of Absorption

Two mechanisms are involved:

1.Simple Diffusion

This is dependent on sugar concentration gradients between the intestinal lumen. Mucosal cells and blood plasma. All the monosaccharides are probably absorbed to some extent by simple ‘passive’ diffusion.

2. “Active “Transport Mechanisms

•Glucose and galactose are absorbed very rapidly and hence it has been suggested that they are absorbed actively and it requires energy.

•Fructose absorption is also rapid but not so much as compared to glucose and galactose but it is definitely faster than pentoses. Hence fructose is not absorbed by simple diffusion alone and it is suggested that some mechanism facilitates its transport, called as” facilitated transport”.

GLYCOLYSIS

Oxidation of glucose or glycogen to pyruvate and lactate is called glycolysis.

This was described by Embeden, Meyerhoff and Parnas. Hence it is also called as Embden Meyerhoff pathway.

It occurs virtually in all tissues. Erythrocytes and nervous tissues derive its energy mainly form glycolysis. This pathway is unique in the sense that it can utilize O2 if available (‘aerobic’) and it can function in absence of O2 also (‘anaerobic’)

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Aerobic Phase

Aerobic phase includes the conversion of glucose to pyruvate

Oxidation is carried out by dehydrogenation and reducing equivalent is transferred to NAD. NADH + H+ in presence of O2 is oxidized in electron- transport chain producing ATP.

•Anaerobic Phase

This phase includes the conversion of Glucose to lactate

NADH cannot be oxidized, so no ATP is produced in electron transport chain. But the NADH is oxidized to NAD+ by conversion of pyruvate to Lactate, without producing ATP.

Anaerobic phase limits the amount of energy per molecule of glucose oxidized. Hence, to provide a given amount of energy, more glucose must undergo glycolysis under anaerobic as compared to aerobic.

A. Enzymes

Enzymes involved in glycolysis are present in cytoplasm.

SIGNIFICANCE OF THE PATHWAY:

•This pathway is meant for provision of energy.

•It has importance in skeletal muscle as glycolysis provides ATP even in absence of O2, muscles can survive under anaerobic condition.

REACTIONS OF GLYCOLYTIC PATHWAY

Series of reactions of glycolytic pathway, which degrades glucose/ glycogen to pyruvate/lactate, are discussed below. For discussion and proper understanding, the various reactions can be arbitrarily divided in to four stages.

Stage I

This is preparatory stage, before the glucose molecule can be split; the rather asymmetric glucose molecule is converted to almost symmetrical form fructose 1, 6 bisphosphate in the presence of ATP.

1. Uptake of Glucose by Cells and its phosphorylation

Glucose is freely permeable to Liver cells. In Intestinal mucosa and kidney tubules, glucose is taken up by ‘active’ transport. In other tissues, like skeletal muscle, cardiac muscle, diaphragm, adipose tissue etc. Insulin facilitates the uptake of glucose. Glucose is then phosphorylated to form glucose – 6- Phosphate. The reaction is catalyzed by the specific enzyme glucokinase in liver cells and by nonspecific Hexokinase in liver and extrahepatic tissues.

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•ATP acts as PO4 donor in the presence of Mg .One high energy PO4 bond is utilized and ADP is produced. The reaction is accompanied by considerable loss of free energy as heat, and hence under physiological conditions is regarded as irreversible.

•Glucose 6 phosphate formed is an important compound at the junction of several metabolic pathways like glycolysis, glycogenesis, glycogenolysis, glyconeogenesis, Hexosemonophosphate Shunt, uronic acid partway. Thus is a “committed step” in metabolic pathways.

2. Conversion of G- 6- phosphate to Fructose6-phosphate

•Glucose6 phosphate after formation is converted to fructose 6-p by phospho- hexose isomerase, which involves an aldose- ketose isomerization. The enzyme can act only on α - anomer of Glucose 6 phosphate.

Phospho- hexose

isomerase

Glucose 6 phosphate Fructose 6 phosphate

3. Conversion of Fructose 6phosphate to Fructose 1, 6 bisphosphate

The above reaction is followed by another phosphorylation. Fructose-6-p is phosphorylated with ATP at 1- position catalyzed by the enzyme phospho- fructokinase-1 to produce the symmetrical molecule fructose –1, 6 bis phosphate.

Note:

•reaction one is irreversible

•One ATP is utilized for phosphorylation of glucose at position 6

•Phosphofruvctokinase I is the key enzyme in glycolysis that regulates the pathway. The enzyme is inducible, as well as allosterically modified

•Phosphofructokinase II is an is enzyme which catalyzes the reaction to form fructose-2 6-

bis phosphate.

Fructose-6-phosphate + ATP Fructose-2, 6-bisphosphate + ADP

Energetics

Note that in this stage glucose oxidation does not yield any useful energy rather there is expenditure of 2 ATP molecules for two phosphorylations (-2 ATP).

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2.The second reaction will produce one ATP. Two molecules of substrate will produce ATP.

+2ATP

Net gain at this stage per molecule of glucose oxidized= + 8ATP

Stage IV

This is the recovery of the PO4 group form 3- phosphoglycerate. The two molecules of 3- phosphoglycerate the end- product of the previous stage, still retains the PO4 group originally derived form ATP in stage 1. Body wants to recover the two ATP spent in first stage for two phosphorylation reactions. This is achieved by following three reactions:

1. Conversion of 3- phosphoglycerate to 2- Phosphoglycerate

3-Phosphoglycerate formed by the above reaction is converted to 2-phosphoglycerate, catalyzed by the enzyme phosphoglycerate mutase. It is likely that 2,3 bisphosphoglycerate is an intermediate in the reaction and probably acts catalytically.

2. Conversion of 2-phosphoglycerate to Phosphoenol pyruvate

The reaction is catalyzed by the enzyme enolase, the enzyme requires the presence of either Mg++ or Mn++ for activity. The reaction involves dehydration and redistribution of energy within the molecule raising the PO4 in position 2 to a “high – energy state”.

3.Conversion of phosphoenol pyruvate to pyruvate

Phosphoenol pyruvate is converted to ‘Enol’ pyruvate, the reaction is catalyzed by the enzyme pyruvate kinase. The high energy PO4 of phosphoenol pyruvate is directly transferred to ADP producing ATP.

Note

•Reaction is irreversible

•ATP is formed at the substrate level without electron transport chain. This is another example of “ substrate level phosphorylation “ in glycolytic pathway

•“Enol“ pyruvate is converted to ‘ Keto’ pyruvate spontaneously.

•But, cells having limited coenzymes, to continue the glycolytic cycle NADH must be oxidized to NAD+-. This is achieved by re oxidation of NADH by conversion of pyruvate to lactate (without producing ATP).

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Significance of lactate formation:

Under anaerobic conditions NADH is re oxidized via lactate formation. This allows glycolysis to proceed in the absence of oxygen. The process generates enough NAD for another cycle of glycolysis.

B.Clinical Importance

•Tissues that function under hypoxic conditions will produce lactic acid from glucose oxidation. Produces local acidosis. If lactate production is more it can produce metabolic acidosis

•Vigorously contracting skeletal muscle will produce lactic acid.

•Whether O2 is present or not, glycolysis in erythrocytes always terminated in to pyruvate and lactate.

Entry of fructose in to glycolysis:

Liver contains specific enzymes fructokinase. It converts fructose to fructose 1 phosphate in the presence of ATP. In liver fructose1-phosphate is split to glyceraldehyde and dihydroxy acetone phosophate by AldolaseB.

Glyceraldehyde enters glycolysis, when it is phosphorylated to glyceraldehyde-3-P by triose kinase.

Dihydroxy aceton phosphate and glyceraldehyde-3-P may be degraded via glycolysis or may be condensed to form glucose by aldolase.

Lack of fructose kinase leads to fructosuria. Absence of aldolaseB leads to hereditary fructose intolerance. If fructose 1, 6 bisphosphatase is absent, causes fructose induced hypoglycemia. The reason being high concentration of Fructose 1 phosphate and fructose 1, 6 bis phosphate inhibit Liver phosphorylase by allosteric modulation.

As in case of Galactose, fructose intolerance can also lead to cataract formation.

Galactose:

Milk sugar contains galactose. Galactokinase converts galatose to galactose-1-P.It reacts with UDP-glucose to form UDP-galactose and glucose-1-P.The enzyme is Galactose-1-P uridyltransferase. UDP-galactose can be epimerized to UDP-glucose by 4- epimerase.Glycogenesis also requires UDP-glucose. UDP-galactose can be condensed with glucose to form lactose.

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Galactosemia:

Some people cannot metabolize galactose. It is an inherited disorder that the defect may be in the galactokinase, uridlyl transferase or 4-epimerase.Most common is uridyl transferase. Such patients have high concentration of Galactose in blood (Galactosemia).In lense, Galactose is reduced to galactitol by aldose reductase.The product accumulates in lense and leads to accumulation of water by osmotic pull. This leads to turbidity of lense proteins (Cataract).

If uridyl transferase was absent galctose 1-phosphate accumulates.Liver is depleted of inorganic phosphate. This ultimately causes failure of liver function and mental retardation.

If 4-epimerase is absent, since the patient can form UDP-galactose from glucose the patient remains symptom free.

Glycogen metabolism

Introduction

Glycogen is the major storage form of carbohydrate in animals .It is mainly stored in liver and muscles and is mobilized as glucose whenever body tissues require.

Degradation of Glycogen (glycogenolysis)

A. Shortening of chains

Golycogen phosphorylase cleaves the α-1, 4 glycosidic bonds between the glucose residues at the non reducing ends of the glycogen by simple phosphorolysis.

•This enzyme contains a molecules of covalently bound pyridoxal phosphate required as a coenzyme,

•Glycogen phosphorylase is a phosphotransferase that sequentially degrades the glycogen

chains at their non reducing ends until four glucose units remain an each chain before a branch point. The resulting structure is called a limit dextrin and phosphorylase cannot degrade it any further. The product of this reaction is Glucose 1 phosphate.

•The glucose 1 phosphate is then converted to glucose 6 phosphate by phosphoglucomutase.

•Conversion of glucose 6 phosphate to glucose occurs in the Liver, Kidney and intestines by the action of Glucose 6 phosphatase. This does not occur in the skeletal muscle as it lacks the Enzyme.

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B. Removal of Branches

A debranching enzyme also called Glucantransferase which contains two activities, Glucantransferase and Glucosidase. The transfer activity removes the terminal 3 glucose residues of one branch and attaches them to a free C4 end of the second branch.The glucose in α-(1,6) linkage at the branch is removed by the action of Glucosidase as free glucose.

C. Lysosomal Degradation of Glycogen

A small amount of glycogen is continuously degraded by the lysosomal enzyme α-(1, 4) glycosidase (acid maltase). The purpose of this pathway is unknown. However, a deficiency of this enzyme causes accumulation of glycogen in vacuoles in the cytosol, resulting in a very serious glycogen storage disease called type II (Pomp’s disease),

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Fig.2.9 Summary of Glycogenolysis

Synthesis of Glygogen (Glycogenesis)

Synthesis of glycogen from Glucose is carried out by the enzyme Glycogen Synthase.The activation of glucose to be used for glycogen synthesis is carried out by the enzyme UDP- glucose pyrophosphorylase. The enzyme exchanges the phosphate on C-1 of glucose-1- phosphate for UDP (Uridinediphosphate). The energy of the phospho glycosyl bond of UDP- glucose is utilized by glycogen Synthase to catalize the incorporation of glucose in to Glycogen. UDP is subsequently released from the enzyme.The α-1,6 branches in glucose are produced by amylo-(1,4-1,6) transglycosylase,also termed as branching enzyme.This enzyme transfers a terminal fragment of 6 to 7 glucose residues(from a polymer of atleast 11 glucose residues long) to an internal glucose residue at the C-6 hydroxyl position.

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Fig 2.10. Glycogenesis

Glycogen storage diseases

These are a group of genetic diseases that result from a defect in an enzyme required for either glycogen synthesis or degradation.They result in either formation of glycogen that has an abnormal structure or the accumulation of excessive amounts of normal glycogen in specific tissues,

A particular enzyme may be defective in a single tissue such as the liver or the defect may be more generalized, affecting muscle, kidney, intestine and myocardium. The severity of the diseases may range from fatal in infancy to mild disorders that are not life threatening some of the more prevalent glycogen storage diseases are the following.

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Table of Glycogen Storage Diseases

Type:	Enzyme Affected	Primary	Manifestations
Name		Organ
Type 0	glycogen synthase	liver	Hypoglycemia and early death,
Type Ia: von	glucose-6-phosphatase	liver	hepatomegaly, kidney
Gierke's			failure,fatty liver,
			hyperlacticacidimia and severe
			hypoglycemia
Type II:	lysosomal a-1,4-	skeletal and	muscular dystrophy, severe
Pompe's	glucosidase, lysosomal acid	cardiac muscle	cardiomegaly,early death.
	a-glucosidase acid maltase
Type V:	muscle phosphorylase	skeletal	Muscle excercise-induced
McArdle's		muscle	cramps and pain, myoglobinuria

The Pentose Phosphate Pathway

The pentose phosphate pathway is primarily an anabolic pathway that utilizes the 6 carbons of glucose to generate 5 carbon sugars and reducing equivalents. However, this pathway does oxidize glucose and under certain conditions can completely oxidize glucose to CO2 and water. The primary functions of this pathway are:

To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells.

To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.

Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates

Enzymes that function primarily in the reductive direction utilize the NADP+/NADPH cofactor pair as co-factors as opposed to oxidative enzymes that utilize the NAD+/NADH cofactor pair. The reactions of fatty acid and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione. The conversion of ribonucleotides to deoxyribonucleotides (through the action of ribonucleotide reductase)

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requires NADPH as the electron source, therefore, any rapidly proliferating cell needs large quantities of NADPH.

Fig 2.11 Oxidative and non –oxidative phases of HMP shunt

Significance of HMP shunt

The net result of the PPP, if not used solely for R5P production, is the oxidation of G6P, a 6 carbon sugar, into a 5 carbon sugar. In turn, 3 moles of 5 carbon sugar are converted, via the

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enzymes of the PPP, back into two moles of 6 carbon sugars and one mole of 3 carbon sugar. The 6 carbon sugars can be recycled into the pathway in the form of G6P, generating more NADPH. The 3 carbon sugar generated is glyceraldehyde-3-phsphate which can be shunted to glycolysis and oxidized to pyruvate. Alternatively, it can be utilized by the gluconeogenic enzymes to generate more 6 carbon sugars (fructose-6-phosphate or glucose-6-phosphate).

Glutathione is the tripeptide γ-glutamylcysteinylglycine. The cysteine thiol plays the role in reducing oxidized thiols in other proteins. Oxidation of 2 cysteine thiols forms a disulfide bond. Although this bond plays a very important role in protein structure and function, inappropriately introduced disulfides can be detrimental. Glutathione can reduce disulfides nonenzymatically. Oxidative stress also generates peroxides that in turn can be reduced by glutathione to generate water and an alcohol. It can also reduce hydrogen per-oxide in to two molecules of water.

Regeneration of reduced glutathione is carried out by the enzyme, glutathione reductase. This enzyme requires the co-factor NADPH when operating in the direction of glutathione reduction which is the thermodynamically favored direction of the reaction.

It should be clear that any disruption in the level of NADPH may have a profound effect upon a cells ability to deal with oxidative stress. No other cell than the erythrocyte is exposed to greater oxidizing conditions. After all it is the oxygen carrier of the body.

The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH. Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival.

Several deficiencies in the level of activity (not function) of glucose-6-phosphate dehydrogenase have been observed to be associated with resistance to the malarial parasite, Plasmodium falciparum, among individuals of Mediterranean and African descent. The basis for this resistance is the weakening of the red cell membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.

Coris Cycle or Lactic Acid Cycle

In an actively contracting muscle, only about 8% of the pyruvate is utilized by the citric acid cycle and the remaining is, therefore, reduced to lactate. The lactic acid thus generated should not be allowed to accumulate in the muscle tissues. The muscle cramps, often associated with strenuous muscular exercise are thought to be due to lactate accumulation. This lactate diffuses into the blood. During exercise, blood lactate level increases considerably. Lactate then reaches

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liver where it is oxidized to pyruvate. It is then taken up through gluconeogenesis pathway and becomes glucose, which can enter into blood and then taken to muscle. This cycle is called cori's cycle, by which the lactate is efficiently reutilized by the body.

Significance of the cycle:

Muscle cannot form glucose by gluconeogenesis process because glucose 6 phosphatase is absent. Unlike Liver, muscle cannot supply Glucose to other organs inspite of having Glycogen.

Fig 2.12 The Cori cycle.

Gluconeogenesis

Gluconoegenesis is the biosynthesis of new glucose from non carbohydrate substrates.

In the absence of dietary intake of carbohydrate liver glycogen can meet these needs for only 10 to 18 hours

During prolonged fast hepatic glycogen stores are depleted and glucose is formed from precursors such as lactate, pyruvate, glycerol and keto acids.

Approximately 90% of gluconeogenesis occurs in the liver whereas kidneys provide 10 % of newly synthesized glucose molecules,

The kidneys thus play a minor role except during prolonged starvation when they become major glucose producing organs.

Reactions Unique to Gluconeogenesis

Seven of the reactions of glycolysis are reversible and are used in the synthesis of glucose from lactate or pyruvate. However three of the reactions are irreversible and must be bypassed by four alternate reactions that energetically favor the synthesis of glucose.

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A. Carboxylation of Pyruvate

In gluconeogenesis, pyruvate is first carboxylated by pyruvate Carboxylase to oxaloacetate (OAA).where it is converted to Phosphoenolpyruvate (PEP) by the action of PEP carboxykinase,

Note: pyruvate carboxylase is found in the mitochondria of liver and kidneys, but not in muscle

1.Biotin is a coenzyme of pyruvate carboxylase derived from vitamin B6 covalently bound to the apoenyme through an ε-amino group of lysine forming the active enzyme.

2.Allosteric regulation

Pyruvate carboxylase is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA may signal one of several metabolic states in which the increased synthesis of oxaloacetate is required. For example, this may occur during starvation where OAA is used for the synthesis of glucose by gluconeogenesis,

At low levels of acetyl COA, pyruvate carboxylase is largely inactive and pyruvate is primarily oxidized in the TCA cycle

B. transport of Oxaloacetate to the Cytosol

Oxaloacetate, formed in mitochondria, must enter the cytosol where the other enzymes of gluconeogenesis are located. However, oxaloacetate is unable to cross the inner mitochondrial membrane directly. It must first be reduced to malate which can then be transported from the mitochondria to the cytosol. In the cytosol, Malate is reoxidized to oxaloactate (see figure 2.13)

C. Decarboxylation of Cytosolic Oxaloacetate.

Oxaloacetate is decarboxylated and phosphorylated in the cytosol by PEP-carboxykinase. The reaction is driven by hydrolysis of GTP

The combined action of pyruvate carboxylase and PEP carboxykinase provides an energetically favorable pathway from pyruvate to PEP.

PEP then enters the reversed reactions of glycolysis until it forms fructose 1, 6- bisphosphate. (see figure 2.13)

D. Dephosphorylation of fructose 1, 6 bisphosphate

Hydrolysis of fructose 1, 6-bisphosphate by fructose 1, 6-bisphosphatase passes the irreversible PFK- 1 reaction and provides energetically favorable pathway for the formation of fructose 6-phosphate.

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This reaction is an important regulatory site of gluconeogenesis,

1. Regulation by energy levels within the cell:

Fructose1, 6 bisphatase is inhibited by elevated levels of AMP, which signal an energy poor state in the cell

Conversely high levels of ATP and low concentrations of AMP stimulate gluconeogensis 2. Regulation by fructose 2,6- bisphoshate

Fructose1, 6-bisphosphatase is inhibited by fructose 2, 6-bisphosphate, an allosteric modifier whose concentration is influenced by the level of circulating glucagons. Fructose 1, 6 bisphos phatase occurs in liver and kidney

E. Dephosphorylation of glucose 6.phosphate

Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase bypasses the irreversible hexokinase reaction provides energetically favorable pathway for the formation of free glucose,

Glucose 6-phosphatase like pyruvate carboxylase, occurs in liver and kidney, but not in muscle.Thus muscle cannot provide blood glucose from muscle glycogen.

C. F. Substrates for Gluconeogenesis

Gluconeogenic precursors are molecules that can give rise to a net synthesis of glucose.They include all the intermediates of glycolysis and the citric acid cycle.

Glycerol, lactate, and the α-keto acids obtained from the deamination of glucogenic amino acids are the most important gluconeogenic precursors.

A.Gluconeogenic Precursors

1.Glycerol is released during hydrolysis of triacylgycerol in adipose tissue and is delivered to the liver. Glycerol is phosphorylated to glycerophosphate an intermediate of glycolysis.

2.Lactate is released in the blood by cells, lacking mitochondria such as red blood cells, and exercising skeletal muscle.

B.Ketogenic compounds

AcetylCoA and compounds that give rise to acetyl CoA (for example acetocetate and ketogenic amino acids) cannot give rise to a net synthesis of glucose, this is due to the irreversible nature of the pyruvate dehydrogenase reaction, (pyruvate to acetyl CoA.) These compounds give rise to ketone bodies and are therefore termed Ketogenic.

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Advantages of Gluconeogenesis

1)Gluconeogenesis meets the requirements of glucose in the body when carbohydrates are not available in sufficient amounts.

2)Regulate Blood glucose level

3)Source of energy for Nervous tissue and Erythrocytes

4)Maintains level of intermediates of TCA cycle

5)Clear the products of metabolism of other tissues(Muscle)

Fig.2.13 Major control mechanisms affecting glycolysis and gluconeogenesis

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Homeostasis of Blood Glucose

Homostasis of glucose is due to balance of addition and utilization of glucose. Fasting blood glucose is maintained between 80-120mg %. After a meal it rises by 40-60mg% and returns to normal within 2-3hours.

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UNIT THREE

INTEGRATIVE METABOLISM AND BIOENERGETICS

Objectives

1.To enable the students:

2.Identify energy rich dietary constituents

3.Identify cellular sites of energy generation

4.Understand mechanism of cellular ATP formation and utilization

5.Understand ways of regulation of cellular energy metabolism

6.Understand the mechanism and effect of poisons on cellular energy generation

Energy Generation and Utilization in the Living System

I-Introduction

Energy is vital to life. Growth, reproduction and tissue repair require energy. Most organisms obtain energy by oxidation of these fuel molecules Carbohydrates, fats and amino acids. Cellular oxidation of these molecules release energy, part of which is conserved through the synthesis of high-energy phosphate bonds and the rest is lost as heat. The high-energy phosphate bonds are directly utilized for cellular energy requiring processes. ATP (adenosine triphosphate) is the common high-energy phosphate bond that is formed during oxidative processes.

Under cellular conditions energy releasing (oxidative) processes are coupled to energy requiring cellular processes through common energy currency, ATP.

It is the universal transfer agent of chemical energy between energy-yielding and energy- requiring cellular processes. Other high energy triphosphates include GTP, UTP, and CTP which are commonly used in biosynthesis (contain comparable energy to that of ATP).

ATP and other nucleotides of comparable energy, carry two high-energy phosphate bonds. The hydrolysis of these high - energy phosphate bonds release energy which powers cellular energy requiring processes. Thioester bonds also contain comparable energy content to that of ATP. Energy of hydrolysis of thioester bond is mostly used to drive the reactions forward to completion.

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II- High-energy phosphate bonds

Fig 3.1 Structure of ATP

a) High-energy phosphate bonds:

- At pH 7, ATP - carries four negative charges

-Charges repel each other because of proximity

-Repulsion is relieved upon hydrolysis of high-energy bonds

ATP and other high energy compounds contain phosphoanhydride bonds which release much free energy upon hydrolysis

b) Energy of hydrolysis of phosphate bonds

hydrolysis of high-energy phosphate bond of ATP releases free energy of

about -7.3 kcal/mol

energy released upon hydrolysis of high energy phosphate bonds may result in:- transfer of phosphate group with partial conservation of energy by newly formed bond formation of new bond

change in conformation of molecules signal amplification

transport molecules across membranes

some portion lost as heat (may contribute to body temperature maintenance in homoeothermic organisms)

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ATP + H2O	ADP + Pi + energy
ATP + Substrate	ADP + substrate – phosphate + heat
Hydrolysis of ATP or other nucleotides usually involves the terminal high-energy		phosphate
bond. Similarly, phosphate transfer	involves the terminal phosphate group. The	phosphate

transfer also commonly involves the two terminal phosphate groups as			pyrophosphate.
Transfer of AMP portion of ATP is also common with concomitant hydrolysis of			pyrophosphate
(energy of hydrolysis driving the reaction forward)
ATP + substrate	Substrate – AMP +PP	2Pi
		heat

C-Cellular formation and utilization of ATP

Within cells ATP is continuously formed and utilized

Serve as the principal immediate donor of free energy in biological system * oxidation of fuel molecules

Catabolism of fuel molecules occurs stepwise each step releasing partial energy content of molecules. The amount of total energy release depends upon the cellular conditions:

i-presence or absence of oxygen (aerobic or anaerobic)

ii-presence or absence of specific organelles with oxidative functions (mitochondrial) Catabolic reactions in addition, provide building blocks for biosynthetic reactions

III- Catabolism of Fuel Molecules –An overview a) Carbohydrates-digestion or mobilization of glycogen

Monosaccharides
Cellular catabolism - Glycolysis	Energy, CO2 and H2O
- Kreb’s cycle
- Electron transport chain
Exception– RBC (red blood cells)
Only glycolysis

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Glycolysis- partial catabolism

-small amount of energy conserved (ATP, NADH)

-prepares carbohydrates for the next catabolic processes

-sometimes the only life sustaining energy generating process

-RBC (red blood cells-lack mitochondrion)

-exercising muscle (oxygen limitation)

b)Fats-digestion or mobilization of stored fat

Fatty acids + glycerol

Transport by albumin

Oxidation by major pathway → β Oxidation

-NADH

-FADH2

-Acety CoA-Common intermediate

No direct high–energy phosphate molecule is formed

c) Proteins-digestion or mobilization of tissue protein
	Glycolytic intermediates
			Further catabolism
Amino acids		acetyl CoA
		Kreb’s cycle intermediates
			Energy and Building blocks

During cellular oxidation of fuel molecules very little energy is directly conserved in the form of high energy phosphate bond that can be directly utilized for cellular energy requiring processes. Most of it is captured in the form of reducing equivalents such as NADH (reduced nicotinamide adenosine dinucleotide) and FADH2 (reduced flavin adenosine dinucleotide).

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Section B- Concept of Free Energy

Definition – Free energy is that portion of the energy of a system available to do work as the system proceeds toward equilibrium under conditions of constant temperature and pressure and volume

The change in free energy content (ΔG) depends on two components

ΔH (change in enthalpy, internal energy heat) and ΔS (change in entropy)

ΔG = ΔH - TΔS

Chemical reaction performs work if it can be harnessed in utilizable form of energy. Amount of work performed depends on the efficiency of the machinery.

Free energy change of a biological reactions is reported as the standard free energy change (ΔG0’)

ΔG0’- is the value of ΔG for a reaction at standard conditions for biological reactions (pH 7, 1M, 25oC, 1 atmosphere pressure)

Free energy change is used to predict the direction and equilibrium of chemical reactions If ΔG is negative – net loss of energy (exergonic)

-reaction goes spontaneously

If ΔG is positive - net gain of energy (endergonic) reaction does not go spontaneously

If ΔG is zero- reactants are in equilibrium

C - Oxidation-Reduction Reactions

The utilization of chemical energy in living system involves oxidation – reduction reactions. For example, the energy of chemical bonds of carbohydrates, lipids and proteins is released and captured in utilization form by processes involving oxidation- reductions.

I- Oxidation - removal of electron(s) from substance

-usually accompanied by a decrease in energy content of oxidized substance II-Reduction - addition of electron(s) to a substance

usually accompanied by an increase in energy content of reduced substance Oxidation reduction reactions are coupled processes

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Example: H2

2e- + 2H+

– first half reaction

–oxidation of H2

–release electrons and protons

–requires second half reaction coupled to

–oxidation of H2

½ O2 + 2e- + 2H+

H2O – second half reaction

–reduction of O2

–oxidation of H2 is coupled to reduction of O2

III- Reduction Potential (Oxidation-reduction potential, E'o )-Concept

Definition - Measure of electron donating tendencies

Electrically measured in reference to a standard substance H2. Determined by measuring the electromotive force generated by a sample half-cell with respect to standard reference half- cell

Anegative E’o	= lower affinity for electrons
A positive E’o	= higher affinity for electrons
H + 2e-		H2	E’o = - 0.42V
½ O2	+	2H+ + 2e-	H2O E’o = + 0.82V

In this example, 2H+/H2 redox pair has negative E’o - Electron donating tendency

where as ½ O2/H2O redox pair has positive E’o

-Electron accepting tendency Hence, H2 - electron donor (oxidized)

O2 - electron acceptor (reduced)

Most molecules serve as both electron donors and electron acceptors of different times depending upon what other substances they react with. In biological systems the primary electron donors are fuel molecules such as carbohydrates, fats and proteins.

The oxidation of these substances transfers electrons to intermediate electron carriers such as NAD+, NADP+ and FAD to reduce them to their reduced state NADH, and FADH2.

Reduction potential of the NAD+ / NADH pair is – 0.32Volt, placed high on the electron tower, good electron donor and that of ½ O2 /H2O is +0.82volts.

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Aerobic Energy-Generation

In aerobic organisms the complete breakdown of fuel molecules, carbohydrates, fats and proteins takes place in mitochondria of eukaryotes and cytoplasmic membrane and cytoplasm of aerobic prokaryotes.

The fuel molecules are metabolized to a common intermediate called aceyl CoA which is further degraded by a common pathway called Kreb’s cycle.

This metabolic pathway in addition to providing energy provides building blocks required for growth, reproduction, repair and maintenance of cellular viability.

Mitochondria - it is an organelle where major amount of energy produced. Structurally it is bounded by two separate membranes (outer mitochondrial membrane and inner mitochondrial

membrane)
Out membrane	- smooth and unfolded
	- Freely permeable to most ions and polar molecules
	(Contain porous channels)
Inner membrane	- folded into cristae-increased surface area

-Highly impermeable to most ions and polar molecules Contain transporters which access polar and ionic molecules in and out Cristae are characteristic of muscle and other metabolically active cell types

-Protein-rich membrane (about 75%)

Inter membrane space – space between outer and inner membranes

Matrix-the internal compartment containing soluble enzymes and mitochondrial genetic material

Fig 3.3 Structure of a mitochondria

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Oxidation of Pyruvate

Pyruvate is common intermediate of many catabolic reactions. It is still energy rich molecule. It is a cross road molecule which can be converted into different intermediates depending on :- type of cells-eukaryotes except RBC- acetyl CoA

-aerobic prokaryotes- acetyl CoA

-anaerobic prokaryotes- ethanol or lactate

-in RBC-always lactate

absence or presence of oxygen -lactate, ethanol or acetyl-CoA

-high ratio of NADH/NAD+ favors lactate formation in actively exercising muscle (oxygen limitation)

Alanine

Oxaloacetate

Pyruvate

Lactate

Acetyl CoA

Oxidation of pyruvate into acetyl CoA-aerobic process (O2 terminal electron-acceptor) which takes place in the mitochondrial matrix of eukaryotic cells Pyruvate is transported into mitochondrial matrix by special transporter.Inside matrix pyruvate is oxidized into acetylCoA by pyruvate dehydrogenase complex which is complex of E1, E2 and E3 enzymes.This enzyme requires s five coenzymes- TPP, Lipoate, CoA, FAD and NAD+

Where:	E1	= pyruvate dehydrogenase,
	E2	= dihydrolipoyl transacetylase,
	E3	= dihydrolipoyl dehydrogenase

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Regulation of Pyruvate Dehydrogenase

Product Inhibition by - acetyCoA

- elevated levels of NADH

Covalent modification - dephosphorylated (active) - Increased ADP/ATP ratio

-Phosphorylated (inactive) - increased acetylCoA/CoA ratio

-Ncreased NADH/NAD+ ratio

KREB’S CYCLE

Also called-tricarboxylic acid cycle (TCA) or Citric acid cycle Final common pathway for complete exudation of carbohydrates, fatty acids and many amino acids.Common pathway for catabolism of acetyl COA ,a common intermediate of different catabolic pathways

Aerobic process (occurs in aerobic cells in presence of oxygen (O2). Reactions take place in cytosol of prokaryotes and mitochondria matrix of eukaryotes

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Reactions of Kreb’s cycle

1.Condensation of acetyl COA with oxaloacetate by citrate synthase (condensing enzyme) to form citrate.

Considerable free energy is lost as heat due to hydrolysis of thisester bond (drive the reaction forward).

2.Isomerization of citrate to isocitrate by aconitase

Aconitase contains iron - sulfer (Fe:S) cluster that assists the enzymatic activity fluoroacetate (potent rodenticide) inhibits aconitase with the ultimate effect of blocking Kreb’s cycle and oxidative phosphorylation.

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3. Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase

There are three types of isocitrate dehydrogenases NAD+ - specific – only mitochondrial NADP+ - specific – cytosolic and mitochondrial

Respiratory chain linked oxidation of isocitrate proceeds almost completely through NAD+ - dependent enzyme

*Cytosolic isocitrate dehydragenase reaction generates NADPH and CO2 anabolic role

4. Oxidative decarboxylation of α- ketoglutarate by α - ketoglutarate dehydrogenase complex

α-ketoglutarate is structurally and functionally similar to pyruvate dehydrogenase complex of three enzymes (A’ B’ C’)

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A’ (α - ketoglutarate dehydrogenase), B’ (transuccinylase), C’ (dihydrolipoyl dehydrogenase). This enzyme has the same coenzyme requirement to that of pyruvate dehydrogenase complex. The reaction releases considerable energy, part of which is used to form high-energy thioester bond and NADH. Arsenite inhibits α-Ketoglutarate dehydrogenese complex blocking Kreb’s cycle. α-Ketoglutarate accumulates upon poisoning of the enzyme. Arsenite is also inhibitor of pyruvate dehydrogenase complex

5.Conversion of succinyl CoA into succinate by succinate thiokinase (succinyl CoA synthetase)

GTP is formed by substrate – level phosphorylation

Fates of GTP - participate in mitochondrial protein synthesis

Converted into ATP.

The ATP thus formed will be transported to the cytosol and used in ATP requiring reactions or converted to other triphosphate nucleotides.

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6. Oxidation of succinate by succinate dehydrogenase

-Stereospecific for transfer of H-atoms of succinate (*)

-Succinate dehydrogenese – is integral part of inner mitochondrial membrane

- Contains Fe – S centers (non-heme iron protein) and FAD as prosthetic groups

Flavoprotein

-Inhibited by malonate which competes for the active site of the enzyme -OOC-CH2-COO- (Malonate)

-Also inhibited by oxaloacetate resulting in succinate accumulation.

7. Hydration of Fumarate by Fumarase

-Fumarase is stereospecific for L-malate (catalyzes stereospecific trans addition of H and OH).

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8.Oxidation of L-malate by NAD–linked malate dehydrogenase

This reaction regenerates oxaloacetate, used in the first reaction.

N.B:

The cycle is aerobic process i.e. regeneration of oxidized coenzymes requires O2 as terminal electron acceptor.

No net consumption or production of cycle intermediates Oxaloacetate plays catalytic role in catabolism of acetyl CoA

Energy of acetyl CoA catabolism is partly conserved as reducing equivalents (NADH and FADH2) and GTP

GTP is formed by substrate – level phosphorylation (synthesis of ATP related to oxidation of substrates not related to electron transport)

Net consumption of two molecules of H2O

Enzymes – soluble and membrane attached (succinate dehydrogenase)

b-Functions of Kreb’s Cycle

Kreb’s cycle has catabolic and anabolic functions

Energy generation

– reducing equivalents,NADH and FADH2

–TP

Provide CO2 used for – gluconeogenesis

–fatty acid synthesis

–urea synthesis

–ucleotide synthesis

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indicating high energy status of the cell. ATP inhibits both citrate synthase and isocitrate dehydrogenase where as both are activated by high levels of ADP. NADH inhibits isocitrate dehydrogenase and α-Ketoglutarate dehydrogenase. Complementary mechanisms of controlling rate of acetyl CoA formation and rate of acetyl CoA degradation is also involved

III- Electron Transport system and Oxidative Phosphorylation

In aerobic organisms the major amount of ATP is synthesized by phosphorylation of ADP related to electron transport, a process occurring on the inner mitochondrial membrane of eukaryotes. It is a system composed of a chain of membrane associated electron carriers.

a- Components:

flavoproteins – FMN and FAD prosthetic groups. Accept H atoms but donate electrons nonheme iron-sulfur proteins (Fe: S centers). Carry electrons not H – atoms. They are component of complexes (I, II and III)

Coenzyme Q - Non protein component, Quinone derivative, lipid soluble. Common form in mammals is Q10 Containing ten isoprene units. Accept H – atom but donate electrons. CoQ is mobile carrier of electrons between flavoproteins and cytochromes. Accept electrons from flavoproteins and donate to cytochromes. Transfer and accept two electrons at a time Cytochromes – heme conjugated proteins

Heme = Fe2+/F3+ + porphyrin

Include classes of cytochromes designated a, b, and c. Iron at the center of cytochromes accept and donates single electron

Cytochrone Fe2+	Cytochrome – Fe3+ + e-
(reduced)	(Oxidized)

Cytochrome with relatively less positive reduction potential (i.e. cyt b) accepts electrons from CoQH2 and transfers them to the next acceptor cytochrome with more positive reduction potential the electron carriers except for CoQ are prosthetic groups of proteins

Components of electron transport system are arranged according to the increasing order reduction potentials, a component with more negative reduction potential – at the top component with more positive reduction potential at the bottom. The components of respiratory chain are organized into four complexes and two mobile electron carriers

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b - Oxidation of reducing equivalents (NADH & FADH2) and electron flow through respiratory chain

NADH	FMN	Reduced Fe –S	CoQ	FADH2
NAD+	FMNH2	Oxidized Fe-S	CoQH2

Complex I

Electrons from NADH enter at complex I to be relayed to CoQ through series of carriers

Q also accepts a pair of electrons from FADH2 prosthetic group of complex II (succinate dehydregenase), glycerol phosphate dehydrogenase (GPDH) and fatty acyl dehydrogenase (FADH)

CoQH2	cytb (Fe3+)	Fe-S (+2)	cytc1 (Fe3+)	cytc (Fe2+)
CoQ	cytb (Fe2+)	Fe-s(+3)	cytc1 (Fe2+)	cytc (Fe3+)

Complex	III
cytc (Fe2+)	cyta (Fe3+)	cyta3	(Fe2+)	Cu (2+)	H2O
cytc (Fe3+)	cyta(Fe2+)	cyta3	(Fe3+)	Cu (1+)	½ O2 + H+

Complex IV

Every step of electron transport in the electron transport chain is accompanied by release of energy

Useful energy is captured at three sites because large enough change in free energy (at least 9 - 10 Kcal/mole) occurs at three different sites when electron pairs flow down from NADH to O2.

The large enough drop in free energy is occurs when electrons are transferred: within the NADH dehydrogenase complex, from NADH to CoQ, within cytochrome reductase from CoQ to Cyt.c, within cytochrome oxidase from cytc to O2. Such drop in free-energy is more than enough to sponsor the synthesis of three ATP from 3ADP and 3PI

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Flow of a pair of electrons from FDH2 to O2 exhibits only two large drops in free-energy, therefore sponsors the synthesis of only two ATP molecules. That is, the drop in free-energy as electrons pass from FADH2 to CoQ is insufficient to sponsor ATP synthesis.

Oxidation of NADH releases more than enough free energy (-52.6kcal) needed for synthesis of 3ATP

Similarly oxidation of FADH2 releases more than enough free energy for synthesis of 2ATP.

C- Coupling of Electron Transport and ATP Synthesis (Oxidative Phosphorylation)

- Electron transport and oxidative phosphorylation are coupled processes

Suggested hypotheses for coupling mechanism

High-energy intermediate serves as precursor of ATP

Activated protein conformation drives the synthesis of ATP

Proton gradient across inner mitochondrial membrane couples electron transport and ATP synthesis (chemiosmostic hypothesis of Peter Mitchell- postulated in 1961)

Mitchell’s chemiosmotic is the accepted theory

According to chemiosmotic theory, electron - transport and oxidative phosphorylation are coupled by proton-gradient as follows. As electrons from NADH or FADH2 flow down the respiratory chain to O2 free energy is released. The free energy released is captured at three sites to pump protons against concentration gradient from matrix to inter membrane space generating proton gradient across inner mitochondrial membrane. As a result of this pH gradient is also formed, more positive (acidic) on the outer side more negative (basic) on the inner side of mitochondria. In this process a proton motive force of 0.22Volts is created across inner mitochondrial membrane. Now the kinetic energy of electrons is transformed into the proton – motive force. This force drives later ATP synthesis

This happens when protons are back translocated into matrix through the channel portion of ATP synthase (Fo) which is activated by electrochemical potential difference

across the membrane. Catalytic portion of ATP synthase (F1) synthesizes ATP from ADP and Pi as protons are back translocated.

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The precise number of protons pumped by each complex is not known with certainty Current estimates - 3 - 4 H + by complex I

-4 H + by complex II

-2 H + by complex IV.

Under cellular conditions, oxidation of: NADH produces 3ATP from 3ADP and 3PI FADH2 produces 2ATP from 2ADP and 2PI Fates of mitochondrial ATP

used by mitochondria energy (ATP) requiring process

translocation to cytosol (where most of ATP is used for biosynthesis) by ATP – ADP translocase (antiporter)

d- Respiratory Control

The most important factor determining the rate of oxidative phosphorylation is the level of ADP which in turn is determined by the rate of ATP consumption cellular energy demand and utilization determines the rate of ATP synthesis through its effect on the level of ADP which controls the rates of oxidative phosphorylation and kreb’s cycle

ATP synthesis requires availability of ADP also regeneration of oxidized coenzymes (NAD+ and FAD) required for Kreb’s cycle requires ADP that can be phosphorylated to ATP

e- Uncoupling of Electron Ttransport and Oxidative Phosphorylation

Uncouplers are substances which uncouple electron transport and oxidative phosphorylation In the presence of uncouplers:

electron transport proceeds

proton translocation by proton pumps proceeds oxygen consumption proceeds

aerobic oxidations proceed without control

Proton gradient is not formed since uncouplers carry protons back to matrix through IMM not through ATP synthase (continuous dissipation of proton – gradient)

ATP synthase not activated No ATP synthesis

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Energy of oxidation lost as heat

Types of Uncouplers:

1.Chemical uncouplers – example 2, 4-dinitrophenol (2, 4-DNP) 2, 4, - DNP accepts proton and carries it into matrix through IMM (membrane permeable)

2.Physiological uncoupler – thermogenin (protein)

H+ -channel in the IMM

Abundant in mitochondria of brown adipose tissue (low level of ATP synthase activity) Responsible for diet induced thermo-genesis

Brown adipose tissue is absent or reduced in obese individuals Present in newborns and cold adapted individuals

Thermogenin is opened by fatty acids liberated upon degradation of stored fat by activated hormone sensitive lipase by norepinephrine released in response to drop in body temperature due to cold environment.

Opening of thermogenin allows reentry of translated protons through IMM (no proton gradient formed

No ATP synthesis

Energy of oxidation lost as heat

biological importance - maintain body temperature in newborns - cold adaptation

f- Respiratory Poisons

Inhibit electron flow through respiratory chain Inhibit proton translocation

Inhibit proton gradient formation Inhibit O2 consumption

Inhibit ATP synthesis

Reducing equivalents remain reduced Other aerobic oxidation’s inhibited

(Kreb’s cycle, pyruvate oxidation, fatty acid oxidation)

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Inhibitor	Complex inhibited
Rotenone	I
Amytal	II
Antimycin A	III
Cyanide (CN-)	IV

Carbon monoxide (CO) , (Azide (N3-) CN- and CO inhibit cytochrome oxidase activity of complex………………………………….. IV

F.Table 3. Inhibition of respiratory chain complexes

ATP synthesis is also inhibited by inhibition of ATP Synthase by oligomycin (blocks Fo portion)

Inhibition of complex IV and ATP Synthase arrest cellular respiration with fatal consequences.

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UNIT FOUR

LIPIDS

Lipids comprise very heterogeneous group of compounds which are insoluble in water but soluble in non-polar organic solvents such us benzene, chloroform, and ether. They are present in all living organisms. The group includes fats, oils, waxes and related compounds.

General Functions of Lipids

i.They are efficient energy sources.

ii.Serve as thermal insulators.

iii.They are structural components of the cell membrane.

iv.Serve as precursors for hormones (steroid hormones).

v.They also dissolve the vitamins, which are fat-soluble and assist their digestion.

Classification: - There are two ways of classification i.e.,

¾Classification as storage and structural lipids and some other functional lipids.

¾Classification based on lipid composition.

I.Simple lipids:- esters of fatty acids with different alcohols. Fats and oils:- These are esters of fatty acids with glycerol.

Waxes:- Esters of fatty acids with high molecular weight monohydric alcohols

II.Complex lipids:- Esters of fatty acids and alcohols together with some other head

groups.

A.Phospholipids:- Esters of the above type containing phosphoric acid residue.

a)Glycerophospholipids:- The alcohol is glycerol

b)Sphingophospholipids:- The alcohol is shingosine.

B.Glycolipids:- Lipids containing fatty acid, sphingosine and carbohydrate residues.

C.Others:- Include sulfolipids, amino lipids and lipoproteins, which are modified forms of lipids.

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III.Derived lipids: include the hydrolytic products of the simple and complex lipids. Eg. Fatty acids, cholesterol etc.

The simplest naturally occurring lipids are triacylglycerols formed by esterification of fatty acids with glycerol. Biological membranes are made up of phospholipids, glycolipids and proteoglycans.

FATTY ACIDS

Fatty acids are building block of most lipids, made of long chain organic acids having one polar carboxyl group (head) and a non-polar hydrocarbon chain (tail). The latter makes them water insoluble. They are not found free in nature but found as esterified forms. Most naturally occurring fatty acids have got even number of carbons. They may be saturated or unsaturated, with one or more double bonds. Mostly the double bond occurs at the 9th carbon as we count from the carboxyl group end.

There are two systems of numbering the carbon atoms in a fatty acid

n	3	2	1
CH3 (CH 2)n CH = CH CH2 CH2 CH2			COOH
ω	β	α

1. Numbering starts from carboxyl carbon. The last carbon is the "n" carbon

2. The second carbon is the "α"and the third the "β" Carbon. The last carbon atom is omega.

Eg:-	CH3	(CH2)7	CH2CH2 (CH2)7 COOH	stearic acid (saturated fatty acid)
Eg:-	CH3	(CH2)7	CH=CH (CH2)7 COOH	oleic acid (Unsaturated fatty acid)

Fatty acids can be represented as shown below where the delta indicates the position of the double bond and the next number shows the number of carbon atoms and the last number indicates the number of double bonds. In a different way the position of the double bond(s) can be indicated as shown in the second expression without the delta.

C18:1, ∆9 or

18:1(9)

C18 indicates 18 carbons, 1 indicates the number of double bonds, delta 9(∆9) indicates the position of double bond between 9th and 10th carbon atoms.

-Double bonds in naturally occuring fatty acids are in the cis- configuration and saturated fatty acids of C12 to C24 are solids at body temperature but the unsaturated once are liquids.

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PUFA (Polyunsaturated fatty acids): They have two or more double bonds .they are called as essential fatty acids because they are required in the body and cannot be synthesized. So they need to be include in the diet.

18	Linoleic acid	18: 2; 9	(12)
18	Linolenic acid	18: 2; 9	(12, 15)
These two are called essential fatty acids.
20	Arachidonic acid	20: 4; (5, 8, 11, 14)

Arachidonic acid is semi essential fatty acid because it can be synthesized from the above two essential fatty acids.

Functions:

1. The fluidity of membrane depends on length and degree of unsaturated fatty acids.

Membrane PL contains essential fatty acids. In case of deficiency of EFA, other fatty acids replace them in the membrane; as a result membrane gets modified structurally and functionally.

2.They are required for the synthesis of PL, cholesterol ester and lipoproteins

3.Poly unsaturated fatty acids are released from membranes, diverted for the synthesis of prostaglandins, leukotriens and thromboxanes.

4.They act as fat mobilizing agents in liver and protect liver from accumulating fats (fatty liver).

TRIACYLGLYCEROLS

These are esters of fatty acids with the alcohol glycerol, which are storage forms of lipids (depot lipids). Triacylglycerols or also called as triacylglycerides, exist as simple or mixed types depending on the type of fatty acids that form esters with the glycerol. Both saturated and/or unsaturated fatty acids can form the ester linkage with the backbone alcohol. Eg. Tripalmitate, Triolein.

Fig 4.1. Structure of Triacylglycerol.R1, R2 and R3 are fatty acids.

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•Tristearin is a chief component of beaf lipid

•Butter has short chain fatty acids.

•Unsaturated fatty acids are sensitive to air and oxidized to give rancid smell.

•Triacylglycerols are mainly found in special cells called adipocytes (fat cells), of the mamary gland, abdomen and under skin of animals.

They produce twice as much energy as that of carbohydrates per gram

STRUCTURE OF LIPIDS

R1 and R2 are fatty acids

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Fig 4.2. Structure of phosphatidate

Phosphatidate is the parent compound for the formation of the different glycerophospholipids. To the phosphate group different head alcohol may be attached. If choline is attached it is called phosphatidyl choline (lecithin), if ethanolamine is attached it is called phosphatidyl ethanolamine.

The second largest membrane lipids are sphingolipids, which contain two non-polar and one polar head groups. Their alcohol is the amino alcohol sphingosine.

Sphingolipids have subclasses viz., sphingomyelins, cerebrosides and gangliosides. Out of these only sphingomyelin contains phosphorus.

CH3 (CH2) 12 CH=CH-CH - CH - CH2OH			Sphingosine
OH NH2

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Sphingomyelins contain as head group phosphocholine or hosphoethanolamine. Below is an example of a sphingomyelin.

CH3(CH2)12 CH=CH –CH - CH-CH2-O-PO3-CH2CH2N(CH3)3

sphingosineOH

CH3(CH2)16CO-NH phosphocholine

Fatty acid

Gangliosides: These are glycolipids most of which are complex containing oligomers of sugars on head groups. One unit shall definitely be N-acetyl neuraminic acid (sialic acid) 6% of grey brain matter is ganglioside.

Cerebrosides:- These are glycolipids which have no phosphate group but neutral head group and contain one or two sugar groups usually glucose or Galactose Functions of phospholipids

1.Phospholipids are components of membrane; impart fluidity and pliability to the membrane.

2.Dipalmitoyl choline (lecithin) acts as surfactant and lowers the surface tension in alveoli of lungs. Lecithin along with sphingomyelin maintains the shape of alveoli and prevents their collapse due to high surface tension of the surrounding medium. Some premature infants can’t secrete lecithin; therefore suffer from respiratory distress syndrome.

3.Intra cellular signals (for second messengers) like inositol triphosphate and diacylglycerol are generated from membrane PL, during the action of hormones.

4.PL anchors certain proteins to cell membranes. PL being amphipathic can interact with nonpolar and polar substances. They link proteins to nonpolar membranes.

5.Solubilization of cholesterol is done by amphipathic nature of PL.

6.Lipids are transported as lipoproteins, which require PL

CHOLESTEROL

•Compounds containing 27 carbon cyclopentanoperhydrophenanthrene structures with four rings labeled A to D. Steroids are complex fat-soluble molecules, which are present in the plasma lipoproteins and outer cell membrane. Cholesterol is one of the important non fatty acid lipid that is grouped with steroids.

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Fig 4.3. Structure of Cholesterol.

Cholesterol is important in many ways:

•For the synthesis of bile salts that are important in lipid digestion and absorption.

•For the synthesis of steroid hormones that are biologically important like the sex hormones estrogen and progesterone.

•For the synthesis of vitamin D3

•As a structural material in biological membranes.

•As a component of lipoproteins as transport forms of lipid based energy.

Digestion and Absorption of Lipids

Diet contains triglycerides, cholesterol and its ester, phospholipids, fattyacids etc.Mouth and gastric juice has got lipase. It can hydrolyse fats without emulsification with bile salts.Milk fat and butter fat is digested by the enzyme.

Major part of fats are digested by pancreatic lipase. It acts on emulsified lipids only. The products are monoglyceride and 2 fatty acids. Monoglyceride is further hydrolyzed by another lipase. Thus 3 fatty acids and one glycerol molecule is produced from the digestion of dietary triglyceride.

Fig 4.4. Action of lipase on TAG

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hospholipids are digested by phospholipases, secreted by pancreas and intestines.

They are four in number, A (A1, A2) B, C, and D. The action of these enzymes are shown down below.

R=Alcohol, R1 and R2 are fatty acids

Fig 4.5: Action of phospholipases on phospholipids

The products of Phospholipase A1 or A2 are Lysophosphosphatidyl choline and one fatty acid, when the substrate is PC.

Phospholipase B acts on Lysophospholipid, produces glycerophosphoryl choline and free fatty acid.

Phospholipase C acts on phospholipids produce diglyceride and Phosphoryl choline.

Phospholipase D acts on phospholipids, produce choline and phosphatidic acid.

Cholesterol esterase hydrolyses cholesterol ester to free cholesterol and one fatty acid. The digested products are water soluble but some are insoluble.

Glycerol, short chain fatty acids enter portal blood directly. Cholesterol, long chain fatty acids are esterified and absorbed in form of micelles .Bile salts are required for the process. Impaired secretion of lipases from the pancreas and bile salts from liver results in failure in fat absorption and causes steatorrea (excessive passage fatty stool). Absorption products of lipid digestion are absorbed from micelles. The micelles, through the intestinal lumen move to the brush border of the mucosal cells where they are absorbed into the intestinal epithelium.

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The bile salts are reabsorbed and reach by enterohepatic circulation to the liver to be used over again. Their absorption is maximum in the ileum and jejunum. The free fatty acids and monoayclglycerols are absorbed through the epithelial cells lining the small intestine and pass to the lymphatic system where they join the systemic blood via the thoracic duct. The intestinal mucosa secretes into the lymph, the absorbed lipids as chylomicrons and VIDL. The former have short life in blood (<lhr) and make plasma milky after rich mea1. The free fatty acids in blood (long chain) are bound to albumin and transported by blood to the liver.

Metabolism of Fatty Acids and Triacylglycerols

The triacylglycerols play an important role in furnishing energy in animals. They have the highest energy content over 9kcal/mole. They provide more than half the energy need of some organs like the brain, liver, heart and resting skeletal muscle.

Mobilization of Fatty Acids from Adipocytes

When the energy supply from diet is limited, the body responds to this deficiency through hormonal signals transmitted to the adipose tissue by release of glucagon, epinephrine, or adrenocorticotropic hormone.

The hormones bind to the plasma membranes of adipocyte cells and stimulate synthesis of cyclicAMP (cAMP). The cAMP activates a protein kinase that phosphorylates and in turn activates hormone-sensitive triacylglycerol lipases (see the mechanism action of Hormones). These lipases hydrolyze the triacylglycerols at position 1 or 3 to produce diacylglycerols (DAG) and fatty acid, which is the rate limiting step in the hydrolysis. The diacylglycerol lipases hydrolyze the DAG to monoacylglycerols (MAG) and a fatty acid. Finally MAG lipases hydrolyze MAG to fatty acid and glycerol.

The free fatty acids (FFA) produced by lipolysis move through the plasma membranes of the adipose cells and endothelial cells of blood capillaries by simple diffusion and bind to albumin in the blood plasma, which are transported to peripheral tissues. The glycerol produced is taken up by liver, phosphorylated and oxidized to dihydroxyacetone phosphate, which is isomerised to glyceraldehydes-3-phosphate, an intermediate of both glycolysis and gluconeogenesis. Therefore, the glycerol is either converted to glucose (gluconeogenesis) or to pyruvate (glycolysis).

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D. Transport of Fatty Acids to the Mitochondria

The fatty acids transported to the different tissue cells must first be activated or primed by reaction with CoenzymeA at the expense of ATP. The reaction is catalyzed by AcylCoA synthetase or also called thiokinase, found in the cytosol and mitochondria of cells. The pyrophosphate generated from ATP favors more Acyl CoA formation by further hydrolysis. In order to undergo β-oxidation, the fatty acids must enter the mitochondria. But they cannot easily cross it as such by passive diffusion.

There are two fatty acid sources viz., those coming from absorption of FFA and those from hydrolysis of triacylglycerols from adipose tissue. The transport of acyl derivatives across the mitochondrial membrane needs three acyltransferases (shuttles).

1.Specific for short chain acyl groups, does not require carnitine

2.Specific for the long chain acyl groups. The shuttles for long chain acyl groups are carnitine acyltransferase I and II. Therefore, long chain acyl groups cross the mitochondrial membrane in combination with carnitine.

Acyl-CoA + Carnitine

Acyl carnitine + CoASH

Enzyme

Fig 4.6. Carnitine transport system.

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The carnitine pools are in the cytosol and mitochondria, abundant in muscle and it is synthesized from the amino acids lysine and methionine in the liver and kidney. The other name of carnitine is β-hydroxy-γ- trimethyl ammonium butyrate. Carnitine acyl transferase I, found in the surface of the outer mitochondrial membrane, catalyzes the acyl transferase reaction from acylCoA to the carnitine. It passes through the outer membrane to the inner membrane of mitochondrion. In the final stage of the transport, the fatty acyl group is released from the carnitine to the intramitochodrial CoASH by carnitine acyltransferase II, which is found in internal surface of the inner mitochondrial membrane. The regenerated acyl CoA is released to the matrix. It is worth noting that acyltransferase I is a regulatory enzyme in β-oxidation. The acyl CoA present in the matrix of the mitochondrion is now ready for β-oxidation.

β-oxidation of Fatty Acids

The successive oxidative removal of two carbons in the form of acetyl–CoA beginning from the carboxyl end is called β-oxidation.It requires a set of enzymes. The oxidation is so called because the β carbon is oxidized during the oxidation process. It takes place in the matrix of mitochondria.

Energy needs of tissues are met by the oxidation of free fatty acids, released by adipose tissue. Fatty acids are activated with the help of thiokinase, prior to transport to mitochondria. Overall activation of fatty acid requires hydrolysis of two phosphodiester bonds.

1.Acyl CoA dehydrogenase converts acyl CoA to acyl trans enoyl CoA

2.Hydratase converts it to 3-hydroxy acyl CoA.

3.Hydroxy acyl CoA dehydrogenase converts it to 3keto acyl CoA.

4.It is further converted to acyl CoA and acetyl CoA.by Thiolase

The cycle is repeated 7 times for palmitic acid for complete oxidation. See the figure

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Fig 4.7: β-oxidation of fatty acid

The FADH2 and NADH +H+ join the electron transport chain as high energy electron carriers. The latter donates its reducing equivalents (hydrogens) to NADH dehydrogenase to produce 3ATP per pair of electrons and the former produces only 2ATPS.

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Complete oxidation of fatty acid can be divided in to two stages.

A. Formation of acetyl CoA.

B. Oxidation of acetyl CoA to CO2, water via TCA cycle.

Stochiometry of the reaction:

Palmitoyl CoA + 7FAD + 7 NAD +7CoA = 8 Acetyl CoA+7FADH2 +7 NADHH.

Energetics of palmitate oxidation:

Reduced equivalents enter ETC and produce energy rich phosphate bonds. Acetyl CoA release energy through TCA cycle.

7 FADH2	→	7 x 2	= 14 ATPs
7NADHH	→	7 x 3	=	21 ATPs
8 Acetyl CoA →		8 x 12 =		96 ATPs

Total ATP produced from one molecule of palmitic acid is 131. Two ATPs (Two energy rich bonds) are utilized, during activation of fatty acid. Therefore total gain of ATPs is 129.

Oxidation of Unsaturated Fatty Acids

The oxidation of unsaturated fatty acids requires two additional enzymes called isomerase and reductase. Most naturally occurring unsaturated fatty acids are in cis- configuration, which are not suitable for the action of enoyl-CoA hydratases and hence they must be changed to their trans isomer by an isomerase. The rest of the enzymes are needed for the oxidation in addition to these two for the oxidation are the same.

Oxidation of Fatty Acids with Odd Number of Carbons

Ruminant animals can oxidize them by B- oxidation producing acetylCoAs until a three carbon propionylCoA residue is left.The acetylCoAs produced are funneled to the Krebs cycle but the propionylCoA produced is converted to succinylCoA by three enzymatic steps. SuccinoyCoA is an intermediate in the Kreb’s cycle and it can be metabolized.

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The fates of acetyl-CoA formed by b-oxidation of fatty acids are:

1.Oxidation to CO2 and H2O by citric acid cycle.

2.Synthesis of lipids like cholesterol, fatty acids and other steroids.

3.Formation of ketone bodies in the liver.

Regulation of Oxidation of Fatty Acids

•Hormones regulate lipolysis, in adipose tissue.More free fatty acids are available for the β- oxidation.

•Insulin inhibits lipolysis.

•Acylcarnitine transferase-1 is inhibited by malonyl CoA , one of the intermediates of fattyacid synthesis.

•High level of NADHH inhibits acyl CoA dehydrogenase.

•Increased concentration of acetyl CoA inhibits Thiolase.

•When the animal is well fed by carbohydrate, fatty acid oxidation is lowered.

The metabolism of Ketone Bodies

When the level of acetyl CoA from β-oxidation increases in excess of that required for entry into the citric acid cycle, It undergoes ketogenesis in the mitochondria of liver (ketone body synthesis). The three compounds viz., acetoacetate, β-hydroxybutyrate, and acetone are collectively known as ketone bodies. The synthesis of ketone bodies takes place during severe starvation or severe diabetes mellitus. During such conditions, the body totally depends on the metabolism of stored triacylglycerols to fulfill its energy demand.

In the synthesis, two molecules of acetyl CoA condense together to form acetoacetyl CoA, a reaction catalyzed by thoilase. Another molecule of acetyl CoA reacts with the acetoacetyl CoA to form 3-Hydroxy-3-methyl glutaryl CoA (HMGCoA). This step is the rate limiting step and the reaction is catalyzed by HMGCoA synthase enzyme. Note that this compound is also an intermediate in the synthesis of cholesterol in the liver cell cytosol but the mitochondrial HMGCoA goes to ketone body synthesis.

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The HMGCoA formed in the hepatocytes mitochondria by the action of the enzyme HMGCoA lyase is changed to acetoacetate.

The acetoacetate, when its concentration is very high in blood is spontaneously decarboxylated to acetone.

Acteoacetate can be converted to β-hydroxy butyrate by a dehydrogenase enzyme. It is a reversible reaction. See the figure

The odor of acetone may be detected in the breath of a person who has a high level of acetoacetate, like diabetic patients. During starvation and severe diabetes mellitus peripheral tissues fully depend on ketone bodies. Even tissues like the heart and brain depend mainly on ketone bodies during such conditions to meet their energy demand.

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Regulation of Ketone Body Synthesis

It is regulated by

•Rate of β-oxidation

•Availablity of substrates to enter TCA cycle

•Mobilization of carbohydrate stores

Utilization of Ketone Bodies

Ketone bodies are produced in the Liver and they are utilized in extrahepatic tissues. Liver does not contain the enzyme required for activation of ketone bodies

Aceto acetate is activated by two processes for its utilization.

1.Aceto acetate + ATP + CoA → Acetoacetyl CoA + AMP. The enzyme is Synthethase

2.Aceteo acetate + Succinyl CoA → Aceto acetyl CoA + Succinate. The enzyme is Thiophorase(Absent in Liver)

Aceto acetyl CoA is broken down to two molecules of Acetyl CoA, which enters TCA cycle for the production of energy.

Aceto acetate and β-hydroxy butyrate are the normal substrates for respiration and important sources of energy .Renal cortex and heart muscle use acetoacetate in preference to glucose

.Brain switches over to utilization of ketone bodies for energy during starvation and in uncontrolled diabetes.

Acetone is exhaled out .It does not produce energy. Normal level of ketone bodies in blood is 1mg %.In ketonemia, the level increases. Excretion of ketone bodies increases in urine, called ketonuria. If the patient suffers from both the signs, it is called ketosis.

Causes of Ketosis:

1.Prolonged starvation, depletion of carbohydrate stores results in increased fatty acid oxidation and ketosis.

2.Lactating mothers develop ketosis, if the carbohydrate demands are not met with.

3.Diabetic patients with uncontrolled blood glucose, invariably suffer from ketosis, ketoacidosis.

Ketosis usually associated with sustained high levels of free fatty acids in blood. Lipolysis and ketogenesis are regulated by hormones.

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In Diabetes, there is lack of insulin, which brings about lipolysis and decreased utilization of glucose.

Lipoysis increases free fatty acids in blood, which are oxidized to meet energy requirements. This causes increased production of acetyl CoA, NADH, ATP which in turn inhibits TCA cycle.

Acetyl CoA requires oxalo acetate to enter TCA cycle .Since oxaloacetate is not forming from glucose, acetyl CoA can’t enter the cycle. It is diverted to ketone bodies synthesis.

Similarly in starvation, due to hypoglycemia, there is less insulin, lipolysis increases and ketogenesis increases .Oxaloacetate is also diverted to gluconeogenesis, which further depletes TCA cycle .So acetyl CoA can only be converted to ketone bodies.

The Biosynthesis of Fatty Acids

Apart from diet fatty acids can be synthesized in the body.

Denovo synthesis of fatty acids take place in cytosol of liver, lactating mammary gland, adipose tissue and renal cortex.

Main site for TG, fatty acid synthesis is adipose tissue.

*Acetyl CoA is converted to Malonyl CoA by acetyl CoA carboxylase.

*Malonyl CoA and acetyl CoA are attached to acyl carrier protein (ACP).

*Malonyl ACP acetyl ACP get condensed to ketoacyl ACP, by condensing enzyme.

*Ketoacyl ACP gets reduced to hydroxyl acyl ACPby a reductase. It requires NADPHH.

*It loses one molecule of water, forms 2-enoyl acyl ACP. Enzyme is dehydratase.

*It undergoes reduction and forms Butyryl ACP.NADPHH and reductase are needed for the reaction.

The formation of malonyl CoA is the committed step in fatty acid synthesis

For the synthesis, all the enzymes are required in the form of fatty acid Synthase complex.

1.Ketoacyl Synthase (condensing enzyme).

2.Ketoacyl reductase

3.Dehydratase

4.Enoyl acyl ACP reductase

5.Thioesterase.

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Acetyl CoA + 7 malonyl CoA + 14NADPHH = Palmitoyl CoA + 7CoA +7CO2 +14 NADP +7H2O.

The main sources of NADPH for fatty acid synthesis are the pentose shunt but the malic enzyme reaction has also a small contribution.

The formation of malonyl CoA is the committed step in fatty acid synthesis

For the synthesis, all the enzymes are required in the form of fatty acid Synthase complex. Stochiometry of the reaction

Acetyl CoA + 7malonyl CoA + 14NADPH + 7H+ → Palmitate + 7CO2 + 14NADP+ + 8CoA + 6H2O

Hence the overall stoichiometry for the synthesis is:

8Acetyl CoA + 7ATP + 14NADPH → Palmitate + 14NADP+ + 8CoA + 6H2O + 7ADP + 7Pi

Regulation o Fatty Acid Synthesis:

•High carbohydrate diet increases synthesis.

•Palmitoyl CoA inhibits synthesis

•Fasting decreases acetyl carboxylase, decreases fatty acid synthesis.

•Insulin stimulates fatty acid synthesis.

Biosynthesis of Triacylglycerols

1.Major pathway: 2.Activate fatty acids are attached to glycerophosphate to form phosphatidic acid ,by acyl transferase.It is converted to diglyceride by the removal of phosphate group by phosphatase.Another fatty acid is attached to the diglyceride to form triglyceride.The synthesis takes place in adipose tissue and Liver.

2.Minor pathway: Monoglyceride is acylated to form diglyceride .It is later converted to triglyceride by addition of one more fatty acid.The process is seen during absorption of Lipids.

Biosynthesis of Cholesterol

Cholesterol is synthesized in the cell cytosol and endoplasmic reticulum from acetylCoA. Liver and intestine account each for 10% of the total cholesterol synthesized in the body. Almost all tissues containing nucleated cells can synthesize cholesterol. The synthesis follows five major steps which include:

•Acetyl CoA is converted to HMG CoA.

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•HMG CoA is reduced to Mevalonate by a reductase.

•Mevalonate undergoes three times Phosphorylation, in the presence of 3 ATPs and various kinases.The product is 3- phosphor-5 pyrophospho mevalonate.

•Dephosphorylation, decarboxylation converts it to Isopentenyl pyrophosphate.

•It is isomerised to dimethyl allyl pyrophosphate by isomeras.

•Isopentenyl pyrophosphate and dimethyl allyl pyrophosphate form Geranyl PP(10C).

•Geranyl PP and one more molecule of Isopentenyl PP→ Farnesyl PP(15C).

•Two of Farnesyl PP join to form Squalene (30C).

1.Squalene undergoes cyclization, loses three carbon atoms,aquire a double bond,forms cholesterol

Regulation of Cholesterol Synthesis:

Acetyl CoA is converted to Mevalonate. It is the committed step in the synthesis of cholesterol.

Almost 800 mg of cholesterol is synthesized in our body .HMG CoA reductase is the regulatory enzyme.

1.Dietary cholesterol inhibits endogenous synthesis.

2.Fasting leads to low levels of the key enzyme.

3.Insulin activates protein phosphatase which converts it to active enzyme. Glucagon decreases its activity through c AMP dependent protein kinase.

4.Whenever ATP levels are low, the enzyme is switched off by AMP activated protein kinase.

5.m RNA for HMG CoA reductase is under the control of sterols . High concentration of sterols inhibits the synthesis of m RNA, there by the synthesis of enzyme.

6.High levels of degradation products lead to rapid degradation of HMG CoA reductase.

Catabolism of Cholesterol:

Intestinal Bacteria converts cholesterol to coprostanol which is excreted in feces. Cholesterol breaks down to cholic acid and chenodeoxycholic acid.Both are bile acids.They combine with sodium, Potassium to form bile salts. The key enzyme α-hydroxylase is inhibited by high concentration of bile acids.

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Functions of Bile Salts

•They lower surface tension ,emulsify fats ,a pre requisite for the action of pancreatic lipase

•They activate Lipase.

•They shift the pH from 9 to 6

•They form micelles with fatty acid,a mono,di,triglyceride and help in absorption

•Promote absorption of fat soluble vitamins

•Bile salts keep cholesterol in soluble form in gall bladder.

•They regulate the breakdown of cholesterol

Cholelitiasis (Gall stones):

Absence of bile salts precipitate cholesterol as gall stones.Solublity of cholesterol depends on the ratio of phospholipids, bile salts to cholesterol.Due to infections bile acids are destroyed which leads to decreases solubility of cholesterol.

Decrease of bile salts can be due to:

A.Failure in enterohepatic circulation

B.Cirrhosis of Liver

C.Disease of ileum.

The patients are treated with chenodeoxycholic acid to solublize the cholesterol or the stones are removed by surgical intervention.

Hypercholesterolemia :

Normal cholesterol level is 150-250mg% in blood.

High concentration leads to hyper cholesterolemia.

Excess cholesterol gets deposited under the skin, tendons as Xanthomas.

In some cases the regulatory enzyme HMG-CoA reductase is not sensitive to feed back regulation. Such people suffer from familial hypercholesterolemia.

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Atherosclerosis:

Deposition of lipids in the connective tissues of intima of arteries is called atherosclerosis. It causes obstruction to blood flow, leading coronary heart disease, stroke, myocardial infarction etc.

The process is initiated when there is injury to endothelial cells of blood vessels. A number of factors are responsible for injury .The condition is compounded by hyper lipidemia.

Atherogenesis is the process by which atherosclerotic plaques form, a critical step in the disease, atherosclerosis.

Low-density lipoprotein complexes (LDLs), which are the primary means of transporting cholesterol in the blood, are readily oxidized.

A class of white blood cells recognizes the oxidation and absorbs the LDL through its scavenger receptor. They becomes engorged and is referred to as a foam cell.

Foam cells attract other white blood cells, which leads to accumulation of more cholesterol.

Ultimately, this accumulation of cholesterol becomes one of the chief chemical constituents of the atherosclerotic plaque that forms at the site.

Circulating monocytes accumulate at the site of injury, ingest excess of lipids.

If the damage to the intima continues, there is infiltration of platelets at the site.

Foam cells and platelets aggregate, and release substances resulting in atheromatic plaque.

Hypercholesterolemic Drugs

•Compactin inhibits HMG CoA reductase.Cholesterol synthesis decreases.

•Mevinolin Competes for Mevalonate ,Cholesterol synthesis decreases

•Cholsetepol, cholesteyramine (Resins) combine with bile salts and inhibit their reabsorption. Here Bile salts are lost in feces. As a result more cholesterol breaks down to bile salts.

•Diatery fiber is not a drug, better than a drug because

•Bile salts gets trapped in fiber and lost in feces.

•More cholesterol breaks down

•Cholesterol absorption is decreased because of indigestible fiber

•All other lipids are absorbed less.

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Lipid Storage Diseases

Lipid storage diseases are also called as sphingolipidosis. They are degraded by hydrolytic enzymes found in lysosomes .When the degradation is impaired; sphingolipids accumulate in the tissues.

1.Nieman-pick disease

Sphingomyelin accumulates in brain, liver and spleen .The condition is due to deficiency of sphingomyelinase .Patient suffers from mental retardation and early death.

2.Gaucher’s disease.

Glucocerebroside accumulates in liver, spleen, brain and bone marrow, due to the deficiency of glucocerebrosidase .Patient suffers from mental retardation.

3.Tay-Sach’s disease.

Hexoseaminidase is absent as a result gangliosides accumulate in brain, spleen and retina. Patient suffers from demyelination. Cerebral degeneration, mental retardation and early death.

Fatty Liver:

Excess accumulation triglycerides in liver causes fatty liver,Liver cirossis and failure of liver function.

Causes are:

•Elivated levels of free fatty acid in blood

•Deficiency of lipotropic factors,which help in the mobilization of fat from liver

•Failure in the secretion of lipoproteins from liver

•Chronic alcoholism

•Prolonged treatment with antibiotics

Lipoproteins

Plasma lipids contain triacylglycerols, cholesterol and other polar lipids.Lipids combined with apolipoproteins to form Lipoproteins. Based on their density they are classified into four subgroups:

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Chylomicrons:

These are derived from intestinal absorption of triacylglycerols and other lipids and have a very short lifespan. They have the least density and richly consist TAG. Chylomicrons transport dietary triacylglycerols and cholesterol from the intestine to the liver for metabolism.

VLDL (very low density lipoproteins):

These are synthesized in the liver and used to transport triacylglycerols from the liver to extrahepatic tissues.

LDL (Low density lipoproteins):

These are produced from the final stage in the catabolism of VLDL. They transport cholesterol synthesized in the liver to peripheral tissues. LDL is metabolized via the LDL- receptorApproximately 30% of the LDL is degraded in extra hepatic tissues, rest is degraded in liver.

HDL (High Density Lipoproteins):

HDL has the highest density in this group since it contains more protein and cholesterol than triacylglycerols. It transports excess cholesterol from peripheral tissues to the liver for degradation and removal. Therefore, HDL cholesterol is good cholesterol but LDL cholesterol is called bad cholesterol.

High concentration of circulating VLDL,LDL are indicative of possible atherosclerosis .Elevated HDL is a good sign which indicates less chances of atherosclerosis .There is a correlation between the incidence of coronary heart disease and low level of HDL.The higher the ratio of HDL/LDL, the less the chances of CHD .

Lipids and Membranes

Membranes are important biological structures, which are indispensable for life. Membranes give cells their individuality by separating them from their surrounding and they are highly selective and semi permeable containing specific gates, pumps, and channels. Membranes control the flow information between cells and their environment since they contain specific receptor molecules in the form of glycoproteins.

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Chemical Composition of Membranes

Phospholipids are the major class of membrane lipids. Cholesterol, glycoproteins and glycolipids are also the other components of membranes. Glycerophospholipids like lecithin, cephalin and phosphatidyl serine. Membranes are mainly formed of phospholipid bilayers. Sphingolipids also form membrane structures, especially that of the brain cells and nerve cells.

Structure of Membranes

All membranes have a bimolecular leaf of lipid bilayers. Proteins are found submerged in the sea of the lipid bilayers (intrinsic proteins) or loosely bound (extrinsic proteins) and cholesterol is also found intercalated between the lipid bilayers giving the fluidy nature of membranes. The integral proteins contain sugar oligomers and most of them function as receptors. Some of the characteristic features of membranes are listed below.

Membranes can be regarded as a sea of lipid bilayers and due to the presence of unsaturated fatty acids and cholesterol. This fluidity enables lateral diffusion of molecules such that integral and non-integral proteins span the whole membrane structure. This implies that membranes are not rigid structures but dynamic structures. The modern representation of lipids as fluidy and dynamic structures is called the fluid mosaic model. The molecules forming membrane structures do not flip-flop or undergo traverse diffusion and therefore, membranes are asymmetric structurally and functionally. The outer and inner surfaces of all known biological membranes have different components and different enzymatic activities.

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UNIT FIVE

AMINO ACIDS

Introduction

There are approximately 300 amino acids present in various animals, plants, and microbial systems, but only 20 amino acids are coded by DNA to appear in proteins.

Cells produce proteins with strikingly different properties and activities by joining the same 20 amino acids in many different combinations and sequences. This indicates that the properties of proteins are determined by the physical and chemical properties of their monomer units, the amino acids.

Definition:

Amino acids are the basic structural units of proteins consisting of an amino group, (-NH2) a carboxyl (-COOH) group a hydrogen (H) atom and a (variable) distinctive (R) group. All of the substituents in amino acid are attached (bonded) to a central α carbon atom. This carbon atom is called α because it is bonded to the carboxyl (acidic) group.

The general formula for the naturally occurring amino acids would be :

H
|α
R – C – COOH
|
NH2	R = variable side chain

Fig 5.1 : General formula of Amino acids

•A basic amino group (-NH2)

•An acidic carboxyl group ( -COOH)

•A hydrogen atom (-H)

•A distinctive side chain (-R)

In neutral solution (PH = 7), both the α- amino and α carboxyl group are ionized resulting the charged form of an amino acids called zwitterion (dipolar) as shown in the figure below.

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COO- |α

NH3+ – C – H

|

R

In dipolar (zwitterion) form the amino group is protonated (-NH3+) and the carboxyl group is dissociated (deprotonated) (-COO-) leading to a net charge zero.

Stereochemistry (Optical activity)

Stereochemistry mainly emphasizes the configuration of amino acids at the α carbon atom, having either D or L- isomers.

COOH	COOH
|	|
H - C – NH2	H2N – C – H
|	|
R	R
D (+) amino acid	L (-) amino acid

Fig 5.2: D and L- forms of Amino acids

Out of the 20 amino acids, proline is not an α amino acid rather an α - imino acid. Except for glycine, all amino acids contain at least one asymmetric carbon atom (the α - carbon atom).

Classification of Amino Acids

L-Amino acids are the building blocks of proteins. They are frequently grouped according to the chemical nature of their side chains. Common groupings of amino acids are aliphatic, hydroxyl/sulfur, cyclic, aromatic, basic, acidic and acid amides. Links to individual amino acids are given below:

I. Structural Classification

This classification is based on the side chain radicals (R-groups) as shown in the table 5.1.Each amino acid is designated by three letter abbreviation eg. Aspartate as Asp and by one letter symbol D.

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II. Electrochemical classification

Amino acids could also be classified based on their acid – base properties Acid amino acids (Negatively charged at pH = 6.0)

Example:

- aspartic acid - glutamic acid

Basic amino acids (positively charged at PH = 6.0)

Example:

- Lysine

- CH2 – CH2 - CH2 - CH2 –NH3+

- Arginine						-
Neutral amino acid
Example:
					Serine	- CH2- OH
	♦
					Threonine	- CH2- OH
	♦
						CH3
					Asparagine	- CH2- CO-NH2
		♦
					Glutamine	- CH2- CH2 - CO-NH2
	♦

E. III. Biological or Physiological Classification

This classification is based on the functional property of amino acids for the organism.

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IV. Classification Based on the Fate of Each Amino acid in Mammals.

Amino acids can be classified here as Glucogenic (potentially be converted to glucose), ketogenic (potentially be converted to ketone bodies) and both glucogenic and ketogenic.

I. Glucogenic Amino Acids

Those amino acids in which their carbon skeleton gets degraded to pyrurate, α ketoglutarate, succinyl CoA, fumrate and oxaloacetate and then converted to Glucose and Glycogen, are called as Glucogenic amino acids.

These include:-

Alanine, cysteine, glycine, Arginine, glutamine, Isoleucine, tyrosine.

II. Ketogenic Amino Acids

Those amino acids in which their carbon skeleton is degraded to Acetoacetyl CoA, or acetyl CoA. then converted to acetone and β-hydroxy butyrate which are the main ketone bodies are called ketogenic amino acids.

These includes:-

Phenylalanine, tyrosine, tryptophan, isoleucine, leucine, and lysine.

These amino acids have ability to form ketone bodies which is particularly evident in untreated diabetes mellitus in which large amounts of ketone bodies are produced by the liver (i.e. not only from fatty acids but also from ketogenic amino acids)

Degradation of Leucine which is an exclusively ketogenic amino acid makes a substantial contribution to ketone bodies during starvation.

III. Ketogenic and glucogenic Amino Acids

The division between ketogenic and glucogenic amino acids is not sharp for amino acids (Tryptophan, phenylalanine, tyrosine and Isoleucine are both ketogenic and glucogenic).

Some of the amino acids that can be converted in to pyruvate, particularly (Alanine, Cysteine and serine, can also potentially form acetoacetate via acetyl CoA especially in severe starvation and untreated diabetes mellitus.

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Fig 5.3. Ketogenic, Glucogenic and Glucogenic-Ketogenic amino acids

Ketogenic and Glucogenic amino acids are as indicated in the chart except Leucine and Lysine which are exclusively ketogenic.

V. Classification Based on Participation in Protein Synthesis.

I. Non-Standard Amino Acids

In addition to the 20 standard amino acids, proteins may contain non- standard (proteogenic) amino acids, which are normally components of proteins but created by modification of the standard amino acids.

Among the non – standard amino acids 4 – hydroxyproline a derivative of proline, 5- hydroxylysine derivative of lysine where both are found in collagen, a fibrous protein of connective tissues. 6 N – methyllysine a constituent of myosin, a contractile protein of muscle and γ-carboxy glutamate a derivative of glutamate, which is found in the blood clotting protein prothrombin.

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				H
			|
	HO – C			CH2
						CH – COO-
	H2C		+
			N
		H		H

4- hydroxyproline

H3N+ - CH2 – CH - CH2 - CH2 – CH - COO-

||

OHNH+3

5 – hydroxylysine

CH3 – NH - CH2 - CH2 - CH2 - CH2 – CH - COO-

|

NH+3

6 – N methyl Lysine

COO-

|

-OOC - CH - CH2 – CH - COO-

|

NH+3

γ– Carboxyglutamate

Fig:5. 4 Non standard amino acids

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II.Non – Proteogenic Amino Acids

These amino acids occur in free or combined state, unlike in proteins,and play important roles in metabolism in plasma, free amino acids are usually found in the order of 10 to 100 μ mol/L, including many that are not found in proteins.

Citrulline ,for example, is an important metabolite of L. arginine and a product of Nitric - Oxide synthase, an enzyme that produces nitric oxide an important signaling molecule.

Antibiotics - gramicidin and antimycin D

γ-aminobutryric acid - which acts as an inhibitory neurotransmitter

D - Alanine - a component of vitamin, panthothenic acid, are some of the non- proteogenic amino acids.

COO-

|

H - C –NH3+

|

CH3

D-Alanine

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Fig 5.5: Non-Proteogenic Amino acids

Ionization States of Amino Acids

Amino acids are amphoteric molecules, that is, they have both basic and acidic groups. Monoamine and monocarboxylic acids are ionized in different ways in solution, depending on the pH of solution.

At pH 7, the “zwitterions” H+3N – CH2 – COO- is the predominant species of Glycine in solution and the overall molecule is electrically neutral. At acidic pH the α amino group (α -NH2 ) group is fully protonated and positively charged, yielding H+3N – CH2 – COOH, while at alkaline pH glycine exists primarily as the anionic H2N – CH2 – COO- species, (Negatively charged species).

At the pH intermediate between pka (a measure of the tendency of group to give up a proton, with that tendency, decreasing 10 fold as the pka increase by one unit) of the amino and carboxyl groups, known as isolectric point (PI), the zwitter ionic form of the amino acid has no net charge.

PI can be calculated for each amino acid with mono amine and mono basic groups as follows: PI: ( ½ PK1 + PK2)

Example : Glycine (G) PKa1 α - carboxyl = 2.4 PKa2 α - amino = 9.8

So, PI = Pka1 + Pka2 2

2.34+9.8 = 5.97 2

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PI can be calculated for Mono amine and dicarboxylic group as follows

Example:- calculate the PI of aspartic Acid (D)

Pka1 α - carboxyl = 2.1
Pka2	α - amino = 9.8
Pka3	R- (side chain) or	- carboxyl group = 3.9
So,	PI = Pka1 + Pka3
	2
	2.1+3.9	= 3
	2

Acid Base Properties of Amino Acids

When a crystalline amino acid, such as Alanine is dissolved in water, it can act as either an acid (proton donor) or a base (proton acceptor)

According to Laury and Bronsted theory of acid and bases, and acid is a proton donor and a base is a proton acceptor.

Example : Alanine acting as proton donor (Acid)

H	H
|	|
H3N+ - C – COO-	H2N – C - COO- + H+
|	|
CH3	CH3
	Net charge (-1)

Alanine acting as a proton acceptor (base)

H

|

H+ + H3N+ - C – COO-	H3N – C - COO- + H+
|	|
CH3	CH3
	Net charge (+1)

Substances having such dual nature are said to be Amphoteric and are often called Ampholytes.

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Titration Curves of Amino Acids

Titration involves the gradual addition or removal of protons.

E.g. Glycine

Each molecule of added base (NaOH) to glycine results in the net removal of one proton. The titration curve plot has two distinctive stages each corresponding to the removal of

from glycine. Each of the two stages resembles in shape the titration curve of monoprotic acid (such as acetic acid).

At very low pH the predominant ionic species of glycine is

H

|

R – C – COOH | + NH3

•The fully protonated form.

At the mid point in the first stages of titration in which the COOH group of glycine loses its proton where equimolar concentrations of proton donor

(H3N+ – CH2 – COOH) and proton acceptor (H3N+ – CH2 – COO-) species are present. At this point pH = pka of the protonated group being titrated for Glycine. The pH at the midpoint is 2.34. Thus its COOH group has a PKa of 2.34.

As the titration proceeds another important point is reached at pH = 5.97 Here, there exists a point of inflection at which removal of the first proton it essentially complete and the removal of the second proton has just begun. At this point glycine exists largely as dipolar ion H3N+ – CH2 – COO- (fully ionized) but with no electric charge. This characteristic pH is called as isoelcetric pH designated as PI or pHI.

So, for glycine, which has no ionizable group as a side chain, the isoelectric point is the Arithmetic mean of the two PKa Values:

PI = ½ (PK1 + pK2)

= ½ (2.34 + 9.60) = 5.97

Glycine will have a net (-negative) charge above its PI and thus moves toward positive electrode (anode) when placed in an electric field.

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At any pH below its PI, glycine has a net positive charge and moves toward negative electrode (cathode, when placed in an electric field.)

So, all amino acids with a single α - amino group and a single α - carboxyl group and an R - group that does not ionize, have titration curves resembling that of glycine.

i.e. PK1 = (the PK of COOH group)

Usually in the range of 1.8 – 2.4

PK2 (of the PK of –NH3 group)

Usually in the range of 8.8 – 11.0

The second stage of the titration curve corresponds to the removal of a proton from the - NH3 group of glycine

The pH at the midpoint of this stage is 9.60 equal to the Pka of – NH+3 group

The titration to the pH of about 12 in which the predominant form of glycine is H2N – CH2 – COO- (negatively charged).

Fig 5. 6: Titration curve of Glycine

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Peptides

G. The peptide bond and its characteristics

Proteins are macromolecules with a backbone formed by polymerization of amino acids in a polyamide structure. These amide bonds in protein, known as peptide bonds formed by linkage of α - carboxyl group of one amino acid with α- amino groups of the next amino acid by amide bonds.

During the formation of a peptide bond, a molecule of water is eliminated as shown below:-

Fig 5.7: Peptide bond synthesis

A peptide chain consisting of two amino acid residues is called a dipeptide, three amino acids tripeptide (e. g Glutathione) etc.

E.g. A tripeptide formed from Cysteine, Glycine and Alanine.

HS H2C	O	O	CH3
|	| |	| |	|

H2N – CH – C - N - CH2 – C – N - CH – COO-

||

HH

Fig 5.8: A tripeptide

By convention, peptide structures are written with amino terminal residues on the left and with the carboxyl terminal residue at the right.

In peptides, the amino acids are joined covalently through peptide bonds, and are formed on partial hydrolysis of much longer polypeptides. Note that the C=O and the NH – bonds are nearly parallel and that the C, O, N, and H are usually co-planar. The C - N single bond in the peptide linkage has ~ 40% double bond character and C = O double bond has ~ 40% single bond character. This has two important consequences.

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1.The imino group (-NH-) of the peptide linkage has no significant tendency to ionize or protonate in the pH range 0 – 14

2.The C - N of a peptide linkage is relatively rigid and cannot rotate freely, a property of supreme importance with respect to the three dimensional conformation of polypeptide chains.

In amide linkage of the peptide bond due to the substantial double bond character there exists little twisting. As a result the group of atoms in the peptide bond exist in the cis or trans nature of the peptide bond. It was found out that the trans configuration is usually favored in order to minimize the steric interaction between bulky R groups on adjacent -carbon atoms. One exception is bonds in the sequence X – Pro, which X is any amino acid followed by Proline. In this case Cis configuration may be favored.

In fact, the peptide bond can be considered a resonance hybrid of the forms

Fig 5.9: A peptide bond

The group of atoms about the peptide bond can exist in either the trans or cis configurations:

Fig 5.10: Tran and cis conformation of peptide bonds

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Peptides of Physiological Significance

Glutathione

Glutathione is a tripeptide formed from amino acids glutamate, cysteine and Glycine, linked together in that order. The glutamate is linked to cysteine through the γ- carboxyl group and α - amino group of cysteine.

Here, the carboxyl group is first activated by ATP to form an acyl-phosphate derivative which is then attacked by cysteine amino group then undergoes condensation with glycine.

Fig 5.11: Glutathione Synthesis

The role of GSH as a reductant is extremely important particularly in the highly oxidizing environment of the erythrocyte. The sulfhydryl of GSH can be used to reduce peroxides formed during oxygen transport. The resulting oxidized form of GSH consists of two molecules disulfide bonded together (abbreviated GSSG). The enzyme glutathione reductase utilizes NADPH as a cofactor to reduce GSSG back to two moles of GSH. Hence, the pentose phosphate pathway is an extremely important pathway of erythrocytes for the continuing production of the NADPH needed by glutathione reductase. In fact as much as 10% of glucose consumption, by erythrocytes, may be mediated by the pentose phosphate pathway.

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Fig5. 12. Structure of GSSG (Glutathione disulphide).

Glutathione is virtually present in all cells often at high levels and can be thought as a kind of redox buffer, which probably helps to maintain.

1)Sulfhydryl groups of proteins in the reduced state

2)Keeps the iron of heme in the ferrious (Fe2+) state

3)Serves as a reducing agent for glutaredoxin.

With its redox function it can also be used to remove toxic peroxides that are formed in the course of growth and metabolism under aerobic condition.

2GSH + R – O – O – H GSSG + H2O + R – OH

Glutathione

Peroxidase

Glutathione peroxidase is a remarkable enzyme that contain a covalently bound selenium (Se) atom in the form of selenocysteine.

Conjugation of drugs by glutathione is often a preliminary reaction catalyzed by cytochrome P450, rendering substances to be more polar and assist their excretion as shown in the figure 5.13.

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PROTEINS

The word protein is derived from Greek word, proteious meaning primary. So, proteins are the major components of any living organism.

Proteins are natural substances with high molecular weights ranging from 5,000 to many millions. Besides Carbon, Hydrogen and Oxygen, they also contain Nitrogen, and sometimes, Sulfur and Phosphorous.

Proteins are most important constituent of cell membranes and cytoplasm. Muscle and blood plasma also contain certain specific proteins.

Protein containing foods are essential for living organism, because protein is the most important biological molecules in building up and maintenances of the structure of body, giving as much energy as carbohydrates in the course of metabolism in the body. Many of the body proteins perform innumerable chemical reactions constantly taking place inside the body.

Proteins are the molecular instruments in which genetic information is expressed; hormones, antibodies, transporters, muscle, the lense protein, antibiotics, mushroom poisons, and a myriad of other substances having distinct biological activities are derived.

Definition

Proteins are macromolecules with a backbone formed by polymerization of amino acids in a polyamide structure.

Classification

Even though there is no universally accepted classification system, proteins may be classified on the basis of their composition, solubility, shape, biological function and on their three dimensional structure.

I. Composition:-

A. Simple protein:

Yields only amino acids and no other major organic or inorganic hydrolysis products i.e. most of the elemental compositions.

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B. Conjugated Proteins

Yields amino acids and other organic and inorganic components

E.g. Nucleoprotein	(a protein containing Nuclei acids)
Lipoprotein	(a protein containing lipids)
Phosphoprotein	(a protein containing phosphorous)
Metalloprotein	(a protein containing metal ions of Fe2+)
Glycoprotein	(a protein containing carbohydrates)

II.Solubility

a)Albumins: These proteins such as egg albumin and serum albumin are readily soluble in water and coagulated by heat.

b)Globulins: these proteins are present in serum, muscle and other tissues and are soluble in dilute salt solution but sparingly in water.

c)Histones:

Histones are present in glandular tissues (thymus, pancreas etc.) soluble in water; they combine with nucleic acids in cells and on hydrolysis yield basic amino acids

III.Overall Shape

A.Fibrous proteins

In these protein, the molecule are constituted by several coiled cross-linked polypeptide chains, they are insoluble in water and highly resistant to enzyme digestion. The ratio of length to breath (axial ratio) is more than 10 in such protein. A few sub groups are listed below.

1.Collagens: the major protein of the connective tissue, insoluble in water, acids or alkalis. But they are convertible to water-soluble gelatin, easily digestible by enzymes.

2.Elastins: present in tendons, arteries and other elastic tissues, not convertible to gelatin.

3.Keratins: protein of hair, nails etc.

B.Globular proteins: These are globular or ovoid in shape, soluble in water and constitute the enzymes, oxygen carrying proteins, hormones etc. the axial ratio is 3 to 4 or less. Subclasses include:- Albumin, globulins and histones.

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IV. On their Biological Functions:

Proteins are sometimes described as the "workhorses" of the cell because they do

So many things Like:
Enzymes:	kinases, transaminases etc.
Storage proteins	myoglobin, ferretin
Regulatory proteins	peptide hormones, DNA binding proteins
Structural protein	collagen, proteoglycan
Protective proteins	blood clotting factors, Immunoglobins,
Transport protein	Hemoglobin, plasma lipoproteins
Contractile or motile Proteins	Actin, tubulin

V. On their level of organization

Primary, secondary, tertiary and quaternary.

a)Primary Structure of Proteins

The primary structure of a protein is defined by the linear sequences of amino acid residues. Protein contain between 50 and 2000 amino acid residues.

The mean molecular mass of an amino acid residue is bout 110 Dalton units (Da). Therefore the molecular mass of most proteins is between 5500 and 220,000 Da. The amino acid composition of a peptide chain has a profound effect on its physical and chemical properties of proteins.

Protein rich in polar amino acids are more water soluble. Proteins rich in aliphatic or aromatic amino groups are relatively insoluble in water and more soluble in cell membranes (can easily cross the cell membrane).

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•The primary structure cannot represent the 3D-nature of a protein molecule since the extended chain of amino acids is co-planar as the covalent bind of peptide is right.

H	O	H	O	H	O	H
⏐	⏐⏐	⏐	⏐⏐	⏐	⏐⏐	⏐

H2N – C – C – N – C – C – N – C – C – N – C – COO-

⏐	⏐	⏐	⏐	⏐	⏐	⏐
R	H	R	H	R	H	R

Fig 5. 15: The primary structure of a protein

b)Secondary Structure

The secondary structure of a protein refers to the local structure of a polypeptide chain, which is determined by Hydrogen bond. The Interactions are between the carbonyl oxygen group of one peptide bond and the amide hydrogen of another near by peptide bond.

There are two types of secondary structure, the ∝ - helix and the β- pleated sheet.

The α - helix

The α - helix is a rod like structure with peptide chains tightly coiled and the side chains of amino acid residues extending outward from the axis of spiral. Each amide carbonyl group is hydrogen bonded to the amide hydrogen of a peptide bond that is 4 - residues away along the same chain. There are 3.6 amino acids residues per turn of the helix the complete turn has 0.54

mmpitch. (1nm = 10-9 m) including nearly 3.6 amino acid residues. This enable every = NH group to bind with a carbonyl O, fourth in line behind the primary structure and the helix winds in a right handed manner in almost all natural protein, i.e. turns in a clockwise fashion around the axis.

Since all the carbonyl oxygen and peptide nitrogen are thus involved in the hydrogen bonds, the hydrophilic nature of the helical region is greatly minimized. As the free energy involved in hydrogen bond is very low, it is formed spontanemsly being weak bonds these are disrupted easily when the chain is extended by a little force and reformed when force is released.

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Fig 5.16. The α helix (a) and the β-pleated sheet (b)

c) Tertiary Structure

The three dimensional, folded and biologically active conformation of a protein is referred to as tertiary structure. The structure reflects the overall shape of the molecule. The three - dimensional tertiary structure of a protein is stabilized by interactions between side. Chain functional group, covalent, disulfide bonds, hydrogen bonds, salt bridges, and hydrophobic interactions.

In the tertiary structure the side chains of Tryptophan and Arginine serve as both hydrogen bond donors and acceptors. Lysine, aspartic acid Glutamic acid, tyrosine and Histidine also can serve as both donors and acceptors in the formation of ion-pairs (salt bridges). Two opposite - charged amino acids, such as glutamate with a γ -carboxyl group and lysine with an ε - amino group, may form a salt bridge, primarily on the surface of proteins.

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d) Quaternary Structure

Quaternary structure refers to a complex or an assembly of two or more separate peptide chains that are held together by non- covalent or, in some case, covalent interactions.

If the subunits are identical, it is a homogeneous quaternary structure; but if there are dissimilarities, it is heterogeneous. For instance insulin consists of A and B chain which are different. Hemoglobin has 4 chains, two of them are α and two are β. these, the polymers may be dimers, trimers, tetramers and so on.

Fig 5.19: Hemoglobin as an example of tertiary structure of a protein

Hemoglobin structure shown above is as an example of quaternary structure of a Protein. Cu, Zn - superoxide dismutase from spinach is a good example of quaternary structure of a protein.

The β- pleated sheet

The β – pleated sheet is an extended structure as opposed the coiled ∝ - helix. It is pleated because the (C-C) bonds are tetrahedral and cannot exist in a planar configuration. If the polypeptide chain runs in the same direction, if forms a parallel β – sheet. It is said to be parallel, and when in opposite direction, antiparallel. A protein molecule may have both type of secondary configuration in different parts of its molecule. Gylcine (Gly) and proline (Pro) residues often occur in β -turns on the surface of globular proteins. Most immunoglobulins have such β-pleated conformation and some enzymes like Hexokinase contain a mixed α-β conformation.

Diagrammatic represntation of hydrogen bonds between -NH and C=O groups on adjacent strands in a β -pleated sheet is as shown above in the Fig 5.16.

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Denaturation of Proteins

Proteins have finite lifetimes. They are also subject to environmental damages like oxidation proteolysis, denaturation and other irreversible modifications.

Denaturation involves the destruction of the higher level structural organization (20, 30 and 40) of protein with the retention of the primary structure by denaturing agents.

A denatured protein loses its native physico-chemical and biological properties since the bonds that stabilize the protein are broken down. Thus the polypeptide chain unfolds itself and remain in solution in the unfolded state. The denatured protein may retain its biological activity by refolding (renaturing) when the denaturing agent is removed.

Fig 5.20: Denaturation of proteins

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Factors that Affect Denaturation

Denaturing agents

1.physical factors

Temperature, pressure, mechanical shear force, ultrasonic vibration and ionizing radiation causes the protein to lose its biological activity.

2.chemical factors

Acids and alkalis, organic solvents (actone, ethanol), detergents (cleaning agents), certain amides urea, guandidine hydrochloride, alkaloids, and heavy metal salts (Hg, Cu, Ba, Zn, Cd…) Cause the denaturation.

Properties of a Denatured Protein

A.an increase in number of reactive and functional group in the composition of the native protein molecule ( side chain group of amino acids, COOH, NH2, SH, OH … etc)

B.Reduced solubility and pronounced propensity for precipitation

this occurs due to loss of the hydration shell and the unfolding of protein molecules with concomitant exposure of hydrophobic radicals and neutralization of charged polar groups.

C.Configurational alteration of the protein molecule.

D.Loss of biological activity evoked by the disarrangement of the native structural molecular organization.

E.Access of proteolytic enzymes in comparasion with the native protein

Clinical Application of Denaturation

The amounts of proteins found in the urine, serum, CSF are utilized to asses various pathological conditions. The appearance of proteins like Albumin and Globulin in the urine can be detected by precipitating them using ammonium sulphate. This could be used to asses the degree of kidney impairment and glomerular permeability.

In some disease, abnormal proteins may be present in plasma and be filtered at the glomerule. The most important member is Bence-jons’ protein which is most often associated with multiple myeloma. So recognition of such protein in the urine may be useful in the diagnosis of the disease.

This could be done by treating few ml of urine with few ml of hydrochloric acid giving a white ring at the junction of the two fluids.

In the case of CSF protein estimation and analysis, a saturated phenol solution is used where 2 drops of CSF with 2ml of 10gm phenol dissolved in distilled water to check for turbidity. If the

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turbidity increases indicates an increase in Globulins. An increase in γ-globulins is observed in case of multiple sclerosis and Neurosyphilis.

The normal level of Albumin in the CSF is 25-30mg%. When CSF treated with sulphosalisalic acid with equal amounts and turbidity increases indicating an increase in the concentration of Albumin in the CSF may be attributed to Acute meningities (usually about 35-40mg% when measured spectrometrically).

Usually the level doesn’t exceed 1gm/L. only in the case of Multiple scleroma and spinal block 10gm/l might be observed.

Hemoglobin

Humans are aerobic organisms. Their lungs extract (O2) from inhaled gases.

The inspired (O2) leads to a more efficient utilization of fatty acids. Where as the expired CO2 is a major product of cellular metabolism.

Living systems contain protein that interact with O2 and consequently increase its solubility in H2O and sequester it for further reaction.

In mammals, Myoglobin (Mb) is found primarily in skeletal and striated muscle which mainly serves as a store of O2 in the cytoplasm and deliver it on demand to the mitochondria.

Where as, Hemoglobin (Hb) is restricted to the Erythrocytes which is responsible for the movement of O2 between lungs and other tissues .

Structure of the Heme Prosthetic Group

Heme is the O2 – binding molecule common to Mb and Hb protophorphyrin IX is the backbone of heme when iron is complexed with protophorphyrin IX it is called Heme. So heme is the prosthetic group in Hemoglobin, Myoglobin and Cytochrome b, c, and c1.

The Fe – porphyrin prosthetic group is, with the exception of two propionate groups, hydrophobic and planar.

Heme become an integral part of the globin proteins during poly peptide synthesis. It is the heme molecule that give globin proteins their characteristic red brown colour. Once the Fe2+ (Ferrious) is incorporated, the protein is called hemoglobin. Such structural coordination creates an environment essential for Globin to bind and release O2.

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If the iron atom were to become oxidized to Fe3+(Ferric), the Globin would get changed to metmyoglobin or (Met hemoglobin) where heme can no longer interact with O2 and O2 transport is compromised.

Fig 5.21: Structure of the heme prosthetic group (protophorphyrin IX) ring system.

Heme is non-covalently bonded in a hydrophobic crevice in the myoglobin and hemoglobin molecules.

Ferrous iron is octahedrally coordinated having six ligands or binding groups, attached to it, the nitrogen atoms account for only four ligands. The two remaining coordination sites which lie along the ring contain on the plane of the ring contains one histidine with imidazole nitrogen that is close enough to bond directly to the Fe2+ called proximal histidine the other histidine which facilitates the alignment of heme to O2 and that of Fe2+ called distal Histidine.Distal histidine confers important geometrical constraints on the six coordination sites which normally restricts the interaction with CO.

The coordinate nitrogen atoms mainly prevents conversion of the heme iron to the ferric state (Fe3+) due to their electron donating character.

In free heme molecules, reaction of oxygen at one of the two “open” coordination bonds of iron which is perpendicular to the plane of the porphryin molecule above and below can result in irreversible conversion of Fe2+ to Fe3+. In heme containing proteins this reaction is prevented by

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sequestering the heme deep within a protein structure where access to the two open coordination bonds is restricted polar amino acids are located almost exclusively on the exterior surface of globin polypeptide and contribute to the high solubility of these proteins. Amino acids which are both polar and hydrophobic, such as Threonine, tyrosine and Tryptophan are oriented to the exterior.

Hydrophobic amino acid residues are buried with in the interior where they stabilize the folding of the polypeptide and binding of iron porphyrin ring.

The only exceptions to this general distribution of amino acids residues in globins are the two Histidines that play an indispensable role in the heme binding are oriented perpendicular to and on either side of the planor heme prosthetic group.

In the quaternary stucture of human Hb there exists two α- globin and two – β- globin sub units (α2 β2). These subunits are arranged in a tetrahedral array. Experimental analysis of the quaternary structure indicates multiple non-convalent interactions between each pair of dissimilar subunits, that is, at the α - β - interfaces. In contrast there are few interactions between identical subunits at the α - α or β – β interface so hemoglobin is considered more as a heterodmer (α β)2. The α β -heterodimer are now recognized as major factors determiners of O2 binding and release.

Myoglobin and Hemoglobin

Both Myoglobin and Hemoglobin are built on a common structural motif. Myoglobin contains a single polypeptide chain folded about a prosthetic group, the heme, which contains the oxygen binding site. Hemoglobin is a tetrameric protein. Each polypeptide subunit closely resembles myoglobin. Note, for example that myoglobin and each subunit of hemoglobin consists of eight helical segments, which are labeled A through H. The multiple subunit structure of hemoglobin gives it important oxygen binding properties that are different than myoglobin's, consistent with hemoglobin's role in oxygen transport. In all vertebrates the oxygen transport protein is hemoglobin, a protein that can pick up oxygen in lungs or gills and deliver it to tissues. Myoglobin, by contrast, is an oxygen storage protein. Oxygen transported to tissues must be released for utilization. In tissues, such as muscle, with high oxygen demands, myoglobin provides large oxygen reserves.

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Adult Hb (HbA)

Contains two types of globin two α - chains (141 residues each) and two β - chains (146 residue each). The amino acid sequences of the two type of subunits are identical at 27 positions.

Fetal Hb (HbF)

Contains a different type of Hb just after conception fetuses synthesize zeta chain (quite like α - chain)

The HbF variant barely detectable and ε- chains just like β - chain later zeta replaced by α - and ε- by γ. HbF contain 2 γ and 2 γ subunits in most adult often increases up to 15 - 20% in individuals with mutant adult Hbs, such as sickle cell disease. This is an example of the body’s compensatory response to a pathologic abnormality .The direct benefit of this structural change in Hb isoform is a more efficient transfer of O2 from maternal HbA to fetal( HbF) .

Sickle Cell Hemoglobin (HbS)

HbS, the variant most commonly associated with sickle cell disease, cannot tolerate high protein concentration when deoxygenated. At low oxygen concentrations, deoxy HbS polymerizes, forms fibers, and distorts erythrocytes in to sickle shapes.

The mutation is Glu6 β -val a surface localized charged amino acids is replaced by a hydrophobic residue as show below

HbA = Val – His – Leu – Thr – Pro – Glu – Glu – Lys

HbS = Val – His – Leu – Thr – Pro – Val – Glu – Lys

Such substitution of Valine (non - polar) for Glutamate (polar) have the following consequence

1.Place A non - polar residue on the outside of HbS which markedly reduce solubility of deoxy HbS. But has little effect on oxy - HbS (causes Hb to clump when deoxygenated)

2.Creates sticky patches on the outside surface of each β - chains (not present HbA)

3.The sticky patches interact with complementary sites of another HbS (oxy) and forms large aggregates that distort the whole RBC structure.

Sickle Cell Trait

The heterozygote individuals (sickle cell trait) (HbA/HbS) is associated with increased resistance to malaria. Specifically growth of the infectious agent, Plasmodium falciparum in the erythrocyte.

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This observation represents an example of a selective advantage that HbA/ HbS heterozygote exhibits over the HbA/HbA normal or the HbS/HbS homozygte.

Sickled erythrocyte exhibits little or less deformity, they no longer move freely through the micorvasculature and often block blood flow. Moreover this cells lose water, become fragile and have a considerably short life span leading to anemia.

Sickle Cell Disease

Sickle cell disease is caused by an inherited structural abnormality in the β –globin polypeptide. Clinically, an individual with sickle cell disease present with intermittent episode of haemolytic and painful vaso–occlusive crisis. The latter leading to severe pain in bone chest and abdomen. There is also a likely to be impaired growth, increased susceptibility to infections and multiple organ damage.

Digestion and Absorption of Proteins

Proteins are larger polypeptide molecules coiled by weaker bonds in their tertiary structure the digestion of proteins involves the gradual breakdown of this polypeptide by enzymatic hydrolysis in to amino acid molecules which are absorbed in the blood stream. The protein load received by the gut is derived from two sources 70-100g dietary protein which is required daily and 35 - 200g endogenous protein (secreted enzymes and proteins in the gut or from intestinal epithelia cell turnover)

Only 1-2g of nitrogen equivalent to 6-12g of proteins are lost in the feces on a daily basis. Thus the digestion and absorption of protein is more efficient.

The process of protein digestion can be divided, depending on the sources of peptidases.

A. Gastric Digestion

Entry of a protein in to stomach stimulates the gastric mucosa to secrete a hormone gastrin which in turn stimulates the secretion of Hcl by the parietal cells of the gastric glands and pepsinogen by the chief cells.

The HCL thus produced lower the pH of stomach to (pH1.5 – 2.5) and acts as an antiseptic and kills most of the bacteria and other foreign cells ingested along with.

The acid denatures the protein and the whole protein susceptible to hydrolysis by the action other proteolytic enzymes.

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Proteases are endopeptidases which attack the internal bonds and liberate large fragments of peptides.

Then pepsinogen having MW 40,000 an inactive precursor or zymogen is converted in to active pepsin in the stomach itself. In this process 44 amino acids gets removed from the amino terminal end and the portion of the molecule that remain intact is enzymatically active pepsin (MW. 33,000).

This active pepsin cleaves the ingested protein at their amino terminus of aromatic amino acids (Phe, Tyr, and Trp.)

The major products of pepsin action are large peptide fragments and some free amino acids.

B. Pancreatic Digestion

Pancreatic zymogens proceed digestion as the acidic stomach contents pass in to the small intestine, A low pH triggers the secretion of a hormone Secretin in the blood.

Secretin stimulates the pancreas to secrete HCO-3 (bicarbonate), which in the small intestine neutralizes the gastric HCL and abruptly change the pH to 7.0.

The entry of large peptide fragments and some free amino acids in the upper part of the small intestine (Duodenum), excites the release of a hormone cholecytokinin (CCK).

CCK:

1)stimulates gall bladder contraction.

2)stimulate secretion of several pancreatic enzymes whose activity is between pH 7and

8 in proenzyme forms.

Three of these pro-enzyme are trypsinogen, chymotrypsinogen and procarboxy peptidase, localized in the exocrine cells.Synthesis of these enzymes as inactive precursors protects the exocrine cells from destructive proteolytic attack.

When the proenzyme reach the lumen of the small intestine, initially the enteropeptidase (old name Enterokinase) a protease produced by duodenal epithelial cells, activates pancreatic trypsinogen to trypsin by the removal of a hexapeptide from NH2 – terminus

Trypsin in turn auto catalytically activates more trypsinogen to trypsin and other proenzymes and liberating chymotrypsin elastas, and carboxypeptidase’s

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These are passed in to the interior of the epithelial cell where other specific peptidases convert almost all of them to a single amino acids that are transported to the blood stream by the opposite side of the cell membrane and carried to liver (primarily) and other tissues for oxidative degradation. This process complete the absorption of 99% of digested proteins. The whole scheme is as shown in the figure below.

Fig 5.22: Digestion and absorption of proteins

II. Transport of Amino Acids in to Intestinal Epithelial cells.

The mechanism of active transport of amino acids are similar with that of glucose uptake.

At the brush - border membrane Na+ - dependent symporters for amino acid uptake are functional with consequent ATP - linked pumping out Na+ at the contraluminal membrane. This is an indirect active transport.

A similar H+ dependent symport is present on the brush border surface of di and tripeptides active transport in to the cell.

Na+ - independent transporters are present on the contralumenal surface, thus allowing amino acids? Facilitated transport to the hepatic portal system.

From both genetic and transporters studies at least six specific symporter systems have been identified for the uptake of L-amino acids from the intestinal lumen.

1.Neutral amino acid symporters with short or polar side chains. Ser, Thr, Ala,

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2.Neutral amino acid symporter for aromatic or hydrophobic side chains. Phe, Tyr,

3.	lmino acid symporter	Pro,and OH – Pro
4.	Basic amino acid symporter	Lys, Arg and Cys.
5.	Acidic amino acid symporter.	Asp, Glu
6.	β amino acid symporter	β-Ala , Tau.

These transporter systems are also present in the renal tubules and defects in their constituent protein structure can lead to disease called Hartnup disease.

Neutral amino Aciduria (Hartnup Disease)

Transport functions, like enzymatic functions, are subject to modification by mutations. An example of a genetic lesion in epithelial amino acid transport is hartnup disease; entry resulting from the defect was first recognized. The disease is characterized by the inability of renal and intestinal epithelial cells to absorb neutral amino acids from the lumen. In the kidney, in which plasma amino acids reach the lumen of the proximal tubule through the Ultra filtrate, the inability to reabsorb amino acids manifests itself as excretion of amino acids in the Urine (aminoaciduria). The intestinal defect results in malabsorption of free amino acids from the diet.

Therefore the clinical symptoms of patients with this are mainly those due to essential amino acid and Nicotinamide deficiencies. The pellagra-like features are explained by a deficiency of Tryptophan, which serves as precursor for nicotinamide. Investigations of patients with Hartnup disease revealed the existence of intestinal transport systems for di - or tripeptides, which are different from the ones for free amino acids. The genetic lesion does not affect transport of peptides, which remains as a pathway for absorption of protein digestion products.

Amino Acid Catabolism

Transamination

The nitrogen component of amino acids, the α - amino groups, must be removed before the carbons can be used in other metabolic pathways. There are several ways that this can be achieved.

The first step in the catabolism of most amino acids is the transfer of their α - amino group to α - ketoglutarate where the products are α - ketoacids and glutamate. This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of transaminases which are also

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called as aminotransferases. Most of the amino acids undergo these reaction except lysine and threonine

Fig 5.23: Transamination of amino acids

Transminase of Clinical Importance

Transaminase is a name for a category of enzymes involved in exchange of an oxygen from an α-keto acid (such as α-ketoglutarate) and an amine from an amino acid

The two most important transminase reactions of high clinical important are Alanine transminase and Aspartate transaminase catalyzed reactions.

Alanine + α-Ketoglutarate <-> Pyruvate + Glutamate

Oxaloacetate + Glutamate <-> Aspartate +-ketoglutarate (Urea cycle)

In addition to their roles as building blocks of proteins, the carbon skeletons may be used to produce energy in oxidative metabolism by the end stages of glycolysis (such as pyruvate from Alanine) and tricarboxylic acid (such as oxaloacetate from Asparate) thereby providing a metabolic fuel for tissues that requre or prefer glucose. In addition, the carbon skeletons of certain amino acids can produce the equivalent of acetyl-CoA or Acetoacetate termed Ketogenic, indicating that they can be metabolized to give immediate precursor of lipids or ketone bodies.

Alanine transaminase (ALT) also called as glutamate pyruvate transaminase (GPT) and Aspartate transaminase (AST) also called as glutamate oxaloacetate transaminase (GOT) are the two most important transaminases of clinical importance. These enzymes are abundant in heart and liver they are released as part of cell injury that occurs in Miocardial infraction (MI), infections hepatitis and damage to either organ. An elevated level of both SGOT and SGPT

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(S=Serum) indicates damage to the Liver. However a rise in SGOT accompanied by only a moderate rise in SGPT suggests damage to heart muscle, skeletal muscle, kidney etc. Assays of these enzyme activities in blood serum can be used both in diagnosis and in monitoring the progress of a patient during treatment.

SGOT: Oxaloacetate + Glutamate <-> Aspartate + -Ketoglutarate

SGPT: Glutamate + Pyruvate -------

> -Ketoglutarate + Alanine

Aminotransferases utilize a coenzyme - pyridoxal phosphate - which is derived from vitamin B6. The functional part of pyridoxal phosphate is an aldehyde functional group attached to a pyridine ring. Catalysis involves a Schiff base intermediate.

Oxidative deamination

Involves the oxidative removal of the amino group, also resulting in ketoacids. the amino acid oxidases are flavoprotein, and produces ammonia.

Amino acid+ FMN + H2O

L-Amino acid oxidase

∝- Ketoacid + FMNH2 + NH3 O2

H2O2 catalase

H2O + O2

FMN

Fig 5.24: oxidative deamination of Amino acids

Dehydration Mechanism

A second means of deamination is possible only for hydroxyamino acids (serine and threonine), through a dehydrates mechanism that involves a dehydration followed by the readdition of water and loss of the amino group as ammonia. Dehydration occurs before deamination and PLP is the prosthetic group.

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