ANATOMY AND PHYSIOLOGY

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Topic One[Cont'd]

Define of terms used in Anatomy and Physiology

BIOCHEMISTRY:
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Topic One: Introduction to Biochemistry
What is Biochemistry?

-         Simplest definition: “Chemistry of the living cell”

-         Uses basic laws of chemistry, biology and physics to explain processes of living cells

-         GOAL: Describe life processes at the molecular level and answer the question:

-         The study of life at the molecular level

Why study biochemistry?

-          Lead us to fundamental understanding of life

-          Understand important issues in medicine, health, and nutrition

Three areas to study:

o Has led to greater molecular understanding of diseases such as diabetes, sickle cell anemia, and cystic fibrosis.
o Next frontier: AIDS, cancer, Alzheimer’s Disease
-          Advance biotechnology industries
o Biotechnology is the application of biological cells, cell components, and biological properties to technically and industrially useful operations

1.         Structural and Functional Biochemistry: Chemical structures and 3D arrangements of molecules.

2.         Informational Biochemistry: Language for storing biological data and for transmitting that data in cells and organisms.

3.         Bioenergetics: The flow of energy in living organisms and how it is transferred from one process to another.

Tools to study biochemistry:

-          Know chemical structures and reactivities of molecules that participate in cellular reactions
-          Know biological function of cellular molecules
- Know how all of the pieces and different pathways fit together
Use knowledge from general chemistry, organic chemistry, and biology and apply it to
biological systems. Concepts and mechanisms are the same.

-    Most biological compounds are made of only

SIX elements: C, H, O, N, P, S
-    Only 31 chemical elements occur naturally in plants and animals
-    All organisms have similar biochemical pathways.
-    All organisms use thensame genetic code.
-   Limited number of molecular building blocks make up larger macromolecules

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Topic 1: Classes of Biomolecules
4 MAJOR CLASSES OF BIOMOLECULES SERVE AS BUILDING BLOCKS FOR LARGER MACROMOLECULES:

1.         Carbohydrates: e.g. glucose, fructose, sucrose
-    mainly used as sources of cellular energy
2.         Lipids: commonly known as fats
-    organic compounds that are not very water soluble
-    used as sources of cellular energy
-    components of cell membranes
3.         Amino Acids:
-    20 natural amino acids in total
-    Used as building blocks for proteins
4.         Nucleotides:
-    5 in total
-    Used as building blocks for DNA and RNA precursors
5.         OTHER:
-    Vitamins: organic compounds necessary for proper growth and development
-           Heme: Organometallic compound containing iron; important for transporting oxygen in your blood stream.
Building blocks are used to create macromolecules: polymer of several, hundreds, to sometimes millions of building blocks

Starch and Cellulose: polymers of glucose molecules that differ only by how the glucose monomers are linked.

-          Proteins/polypeptides: amino acid monomers linked together

-          DNA:deoxyribonucleic acid
o          Heteropolymer of monomeric nucleotides
o    Storage of genetic information

-    RNA: ribonucleic acid
o          Heteropolymer of monomeric nucleotides
o          Involved in the TRANSFER of the genetic information encoded by DNA

Biomacromolecules:

-          self-assemble into cellular structures and complexes.

-          recognize and interact with one another in specific ways to perform essential cellular

functions (e.g. membranes are complexes of lipids and proteins)

-          Interactions are weak and reversible

-          Molecules have three dimensions and shapes! Much of biochemistry relies on this fact.

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Topic 1: Chemical Bonding: Ionic Bonding
Chemical Bonding

Octet rule-Duet role: when undergoing chemical reaction, atoms of group 1A-7A elements tend to gain, lose, or share sufficient electrons to achieve an electron configuration having eight valance electrons. After gaining, losing, or sharing, ions/atoms will have the electron configuration like that of the noble gases nearest to them in atomic number.

Note: for the main-group elements (1A-7A), the first level can have a maximum 2 electrons

(for hydrogen and helium) and other levels 8 electrons.

Metal (usually): loses one, two or three electrons and in losing electrons, the atom becomes a positively charged ion called a Cation (Na+, Ca2+, Mg2+). Cation has an electron configuration like that of the noble gas nearest to it in atomic number.

Nonmetal (usually): gains one, two or three electrons and in gaining electrons, the atom becomes a negatively charged ion called an Anion (Cl-, O2-, S2-). Anion has an electron configuration like that of the noble gas nearest to it in atomic number.

Note: the octet rule is not perfect for two reasons:

1.   Ions of period 1 and 2 elements with charges greater than +2 are unstable (B3+, C4+ and C4- don’t exist because they are unstable). It is far too large a charge for an ion of these period elements.

2.   The octet rule does not apply to the type II cations (most of the transition and inner transition elements, groups 1B-7B).

Naming Monatomic cations: name of metal + “ion”

Na+        Sodium ion     Ba2+      Barium ion     Al3+       Aluminium ion

If elements have more than one type of cation (most transition and inner transition elements), we show the charge with the Roman numeral immediately following the name of the metal (for Systematic name or IUPAC (International Union of Pure and Applied Chemistry)). We can also use the suffix “-ous” to show the smaller charge and the suffix “-ic” to show the larger charge (for Common name).

Cu+     Copper(I) ion Cuprous ion

Cu2+   Copper(II) ion Cupric ion

Naming Monatomic anions: we add “-ide” to the step part of the name.

F-         fluor    Fluoride

Cl-       chlor    Chloride

Ionic bonds: ionic bonds usually form between a metal and a nonmetal. In ionic bonding, electrons are completely transferred from one atom to another. In the process of either losing or gaining negatively charged electrons, the reacting atoms form ions. The oppositely charged ions are attracted to each other by electrostatic forces, which are the basis of the ionic bond.

Na (1s2 2s2 2p6 3s1) + Cl (1s2 2s2 2p6 3s1 3p5) → Na+ (1s2 2s2 2p6) + Cl- (1s2 2s2 2p6 3s1 3p6)

Note: there are enormous differences between the chemical and physical properties of an atom and those of its ion(s). For example sodium is a soft metal and it reacts violently with water. Chlorine is a gas and it is very unstable and reactive. Both sodium and chlorine are poisonous. However, NaCl (common table salt made up of Na+ and Cl-) is quite stable and unreactive.

Note: the charge of a monatomic ion can be predicted from the periodic table:

Group # Valence e-    # Gained/Lost Charge on Ion

1A       1          1          +1

2A       2          2          +2

3A       3          3          +3

5A       5          3          -3

6A       6          2          -2

7A       7          1          -1

8A       8          0          0

Note: matters are electrically neutral (uncharged). The total number of positive charges must equal the total number of negative charges. The subscripts in the formulas for ionic compounds represent the ratio of the ions.

Na+        Cl-   →   NaCl             Ca2+      Cl-   →   CaCl2                  Al3+       S2-   →   Al2S3

Ba2+      O2- → Ba2O2                   we must reduce to lowest terms:       BaO

Naming binary ionic compounds: Name of cation (metal) + name of anion

Note: We generally ignore subscripts in naming binary ionic compounds.

Note: many transition metals form more than one positive ion. We use Roman numerals in the name to show their charges.

NaCl   Sodium chloride         CaO    Calcium oxide            AlCl3 Aluminium chloride CuO    Copper(II) oxide (cupric oxide)        FeCl2    Iron(II) chloride (Ferrous chloride) MgCO3                 Magnesium carbonate            NaOH             Sodium hydroxide

Covalent bonds: covalent bonds usually form between two nonmetals or a metalloid and a nonmetal. In covalent bonds, the atoms share one or more pairs of electrons (by using their valence electrons) between each other to obtain a filled valence level.

Note: the valence electrons which are shared between two atoms are called “shared pair of electrons” or “bonding pair of electrons”. The valance electrons which are not shared are called “unshared pair of electrons” or “lone pairs”. These electrons are not involved in bonding.

Note: Only valance electrons are involved in a chemical bond and/or a chemical reaction. The core electrons located in other levels (inside levels) are not involved.

Electronegativity: electronegativity is measure of an atom’s attraction for the electrons it shares in a chemical bond with another atom. The electronegativity shows us how tightly an atom holds the electrons that it shares with another atom.

Note: electronegativity generally increases from left to right across a row of the Periodic Table.

Note: electronegativity generally increases from bottom to top within a column of the Periodic Table.

Note: when the electronegativity increases, the ionisation energy increases too. We classify covalent bonds into two categories:

1. Nonpolar covalent bonds: bonding with an equal sharing of electrons.

H-H                 Cl-Cl               N-N

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Topic 1: Chemical Bonding
2. Polar covalent bonds: bonding with an unequal sharing of electrons. The number of shared electrons depends on the number of electrons needed to complete the octet.

O-H                 O-F                 N-H

The less electronegative atom has a lesser fraction of the shared electrons and obtains a partial positive charge (δ+). The more electronegative atom gains a greater fraction of the shared electrons and obtains a partial negative charge (δ-). This separation of charge produces a dipole (two poles). We show a bond dipole by an arrow, with the head of the arrow near the negative end of the dipole and a cross on the tail of the arrow.

δ+    δ-

H - Cl

Note: we use the following table to find the type of a chemical bond:

Electronegativity difference between bonded atoms Type of bond

less than 0.5    nonpolar covalent

0.5 to 1.9         polar covalent

greater than 1.9           Ionic

Examples:

H-Cl

3.0 - 2.1 = 0.9

Polar covalent bond

C-H

Zn-O   2.5 - 2.1 = 0.4

3.5 - 1.6 = 1.9 Nonpolar covalent bond

Ionic bond

Covalent compounds: the most common method to show the covalent compound is Lewis Structure. First, we should determine the number of valence electrons in the molecules. Then, we connect the atoms by single bonds. Finally, we arrange the remaining electrons so that each atom has a complete outer level.

Resonance: a molecule shows resonance when more than one Lewis structure can be drawn for the molecule. In such a case we call the various Lewis structures Resonance Structures.

Naming binary covalent compounds: name the less electronegative element (the first element in the formula) + name the more electronegative element (the second element in the formula) + adding “-ide” to the stem part of the name. We use the prefixes mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca- to show the number of atoms of each element.

Note: the prefix “mono-” is omitted for the first atom named and it is rarely used with the second atom.

Note: we drop “a” when following a vowel.

SO3         Sulfur trioxide        PCl5    Phosphorus pentachloride

N2O4 Dinitrogen tetroxide   OF2     Oxygen difluoride

Bond angle (VSEPR model) and geometric structure: the angle between two atoms bonded to a central atom. According to VSEPR model, the valance electrons of an atom may be

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Topic Two: Protein synthesis
Protein is a polymer of amino acids joined together by peptide bonds. In the process of protein synthesis also known as translation of m-RNA, the amino acids are added sequentially in a specific number and sequence, determined by the sequence of codons in the genetic code of the relevant m-RNA.

Materials Required for Protein Synthesis

• Amino acids—at least 20 amino acids

• DNA and three RNAs—m-RNA, t-RNA and r-RNA

• Polyribosomes (Polysomes)

• Enzymes – Amino-acyl-t-RNA synthetase : enzyme required for activation of amino acids. – Peptide synthetase (Peptidyl transferase)

• Factors – Initiation factors— eIF-1, eIF-2, eIF-3 eIF-4A, eIF-4B, eIF-4G eIF-4E, eIF-5 – Elongation factors—EF1 and EF2 – Release factors R1 and R2

• Coenzymes and Cofactors – F.H 4 —required in prokaryotes only for formylation of methionine – Mg ++

• Energy: ATP and GTP.
Protein synthesis takes place on ribosomes which is a nucleoprotein and contains 65 per cent r-RNA and 35 per cent proteins. It is a large particle and in prokaryotes it has 70S as its sedimentation coefficient while 80S in eukaryotes

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Topic Two: Steps in Protein synthesis
STEPS OF PROTEIN SYNTHESIS

The process of protein synthesis (after transcription has taken place) can be divided in following steps:

1. Activation of amino acids,

2. Initiation,

3. Elongation, and

4. Termination.

1. Activation of amino acids: (formation of aminoacyl t-RNA)

The amino acids need to be activated before they can be incorporated into the peptide chain. The key enzyme in this process is aminoacyl t-RNA synthetase. These are specific for a particular L-amino acid and also for t-RNA.

2. Initiation The initiation may be divided arbitrarily into following 4 steps:

A. Dissociation of the ribosome 80S into 60S and 40S subunits.

B. Formation of 43S preinitiation complex

C. Formation of initiation complex

D. Formation of 80S initiation complex

3. Elongation

• Elongation is a cyclic process on the ribosome in which one amino acid is added to the nascent peptide chain.

• The peptide sequence is determined by the codons present in the m-RNA.

• It requires elongation factors—EF-IA, EF-2. Steps involved in elongation:

The steps are mainly three:

A. The binding of new aminoacyl-tRNA to ‘A’ site

B. Peptide bond formation

C. Translocation process.

4. Termination Process

After multiple cycles of elongation process, it results to formation of polypeptide chain. When the desired protein molecule is synthesised, a stop codon or terminating codon appears in the A site of m-RNA. The stop codons are: UAA, UAG, or UGA. There is no t-RNA with an anticodon capable of recognising such a termination signal. Releasing factor RF-1 recognises that a stop codon has come in the ‘A’ site. This protein factor RF-1 is a complex consisting of another releasing factor RF-3 with bound GTP. This complex with the help of “peptidyl transferase” brings about hydrolysis of the bond between the peptide and the t-RNA occupying the ‘P’ site. The

Energy is provided by GTP → GDP + Pi conversion. The hydrolysis releases the synthesised peptide chain, m-RNA and t-RNA from the ‘P’ site. The 80S ribosome now dissociates into 60S and 40S subunits which are then recycled.

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Topic 1: Water
Objectives
The learner to be able to explain the structure, properties and function of water

While modern biochemistry tends to focus on the structure and function of molecules such as proteins and DNA, it is important to keep in mind that biomolecular structure and function are dictated by the properties of the medium in which they are dissolved.

Therefore, this chapter presents an overview of the properties of water that are germane to the structure and function of biomolecules.

As an illustration of the importance of water in biological systems, consider the formation of biological membranes.

Cell membranes ultimately form due to the fact that the acyl chains of glycerophospholipids are not soluble in water.

As a consequence, glycerophospholipids and other membrane lipids cluster together leading to structures such as the cytoplasmic membrane and membranes of organelles.

In this Section, we will review fundamental properties of water such as solvation of polar and nonpolar molecules, water ionization and pH, and acid-base chemistry and buffering systems.

These topics are essential for understanding everything that will be discussed in later chapters

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Topic 1: properties of water molecules
II. General properties of water molecules.

The oxygen atom in a water molecule has an sp3 arrangement of bonding orbitals in which the 2 H atoms and 2 unshared pairs of electrons are located in a tetrahedral arrangement around the oxygen.

This arrangement results in a net dipole in which the end of the molecule containing the unshared electrons has partial negative character and the end containing the 2 hydrogens has partial positive character

In addition, each H-O- bond also has dipolar character due to unequal sharing of electrons between hydrogen and oxygen.

Due to the fact that a net dipole exists in individual water molecules, water is regarded as a polar solvent. The polarity of other small molecules is considered

III. H-bonding in water.

Neighboring molecules in bulk water are held together by non-covalent bonds known as H-bonds. The configuration of atoms in an H-bond is illustrated in.

The H- bond is the non-covalent attraction (dashed line) between the partially positively charged H atom attached to the left oxygen atom and one of the unshared electron pairs (not shown) of the oxygen atom on the right.

Each water molecule has 2 unshared electron pairs and 2 hydrogens that can participate in H-bonding. Thus, each water molecule can form H-bond to 4 neighbors.

Since sp3 molecular orbitals are tetrahedrally oriented, neighboring water molecules surrounding a given water molecule are located in a tetrahedral arrangement.

In ice, water molecules are organized in a rigid, precisely tetrahedral crystalline lattice, where each molecule is H-bonded to 4 others.

In liquid water, each water molecule is H-bonded to ~3.4 others on average. Local groups, i.e., "flickering clusters," of molecules only exist for nanoseconds.

While a roughly tetrahedral arrangement of molecules is present, liquid water is more dense than ice because the somewhat irregular packing of molecules allows them to fit together a bit closer.

Due to extensive H-bonding, water is highly cohesive.

The cohesiveness of molecules confers a high melting point and boiling point in spite of the low molecular weight of water (18 g/mol).

The high specific heat and heat of evaporation make water an excellent thermal buffer for actively metabolizing cells and tissues.

It also explains why cold water can quickly conduct heat away from a swimmer leading to hypothermia and possibly death.

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Topic 1: Properties of Water Cont... 1

IV. Behavior of ionic and polar substances in water.

Because water molecules are polar, ionic compounds (electrolytes) and polar molecules are relatively soluble in water.

Substances that can dissolve readily in water are referred to as hydrophilic. For salts (e.g., NaCl), both the cationic (Na+) and anionic (Cl-) components of the salt can be solvated via interactions with the negative and positive, respectively, ends of dipolar water molecules.

Because the interactions are energetically favorable, the salt dissolves.

Dissolved ions are considered to be "solvated" or "hydrated."

The shells of surrounding water molecules shield the ions preventing them from strongly interacting and reforming the crystal.

Water molecules also form H-bonds to polar functional groups in polar biomolecules such as sugars and amino acids.

The different types of H-bonds that can form are discussed below.

It should be noted that many biomolecules contain a combination of polar and nonpolar groups. Thus, the actual solubility of biomolecules is quite variable and depends on the relative proportions of polar and nonpolar regions.

V. Behavior of nonpolar substances.

Nonpolar substances are relatively insoluble in water and therefore are referred to as hydrophobic.

Such molecules typically are hydrocarbons containing methylene, methyl, and aromatic ring functional groups.

They generally lack polar groups that can interact with water molecules.

Because water molecules cannot form H-bonds to a nonpolar substance, water molecules become highly ordered in the immediate vicinity of the compound forming ice-like bonds to one another.

Indeed cage-like structures known as clathrates are formed which can be viewed as rigid geodesic domes surrounding the nonpolar molecule.

All this structuring decreases the disorder or entropy of the water, which is an energetically unfavorable process.

To avoid this situation as much as possible, the suspended hydrophobic substances coalesce which reduces the surface area of the nonpolar molecules in contact with water.

The term hydrophobic interactions refers to the clustering together of nonpolar molecules such as membrane lipids to avoid the entropically unfavorable process of ordering neighboring water molecules.

It is important to note that hydrophobic interactions are not a type of chemical bond per se.

One other important class of molecules--the amphiphiles--deserves mention.

These molecules have significant proportions of both hydrophilic and hydrophobic functional groups.

Typical examples are detergents such as sodium dodecyl sulfate (SDS) which contains a highly water soluble sulfate group and a very insoluble 12-carbon alkyl group.

This schizophrenic combination results in the hydrocarbon chains clustering together away from water contact when SDS is added to water.

In this case, the clusters formed are spherical structures known as micelle.

At an air-water surface SDS molecules actually line up with their hydrocarbon tails pointing up into the air and the sulfate groups in contact with water.

SDS is a useful detergent. Its hydrocarbon tail will bind to nonpolar surfaces, such as greasy dirt, and dissolve it within the interior of the micelle.

After the dirt-filled micelles are suspended in water by agitation, the dirt and detergent can be rinsed away.

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Topic 1: Properties of Water Cont... 2
VI. Noncovalent interactions in biomolecules.

Weak, reversible bonds (noncovalent bonds or noncovalent interactions) mediate interactions between biomolecules. Noncovalent bonds are "individually weak, but collectively strong" and together stabilize the complex structures of biomolecules such as proteins.

However, because they are individually weak, biomolecules exhibit flexibility which is important in processes such as enzyme catalysis.

Furthermore, non-covalent interactions allow reversible binding of small biomolecules to enzymes and nucleic acids. Generally, noncovalent interactions are less than 1/10 th as strong as covalent bonds such as the -C-H bond.

A. Charge-charge interactions.

Charge-charge interactions occur between oppositely charged functional groups or ions.

These bonds are also known as ion pairing interactions and salt-bridges.

The strength of these bonds is inversely dependent on the square of the distance separating the charges.

Strength also depends on the medium in which they occur, with polar media such as water weakening interactions through solvation of interacting ions.

Repulsive forces between like charges also can play an important role in biological processes.

B. H-bonds.

The H-bonds that occur between water molecules are just one example of the many types found in biomolecules.

In general, an H-bond is defined as a dipolar attraction between the hydrogen atom attached to one electronegative atom, and a second electronegative atom.

The H atom must be covalently bonded to an electronegative atom such as O or N to generate a molecular dipole.

The atom with the covalently bound hydrogen atom is called the hydrogen donor, and the other atom is the hydrogen acceptor.

The distance between the two electronegative atoms in an H-bond is ~0.3 nm (3 Å).

H-bond strength is highly dependent on the alignment of molecular orbitals in the interacting molecules and is strongest when they are lined up properly.

As a result, H-bonds are very important in establishing specificity in molecular interactions, e.g., A-T and G-C base pairing in DNA.

C. van der Waals forces.

These forces are attractions between oppositely oriented dipoles that are transiently induced in the electron clouds of closely interacting molecules.

The strength of these forces is maximal when the interacting molecules are just touching.

In fact, these forces become destabilizing and push molecules apart if molecules are compressed more tightly together.

Note that the van der Waals contact radius is defined as the distance at which the attraction of molecules is maximal. van der Waals forces typically are the weakest of the noncovalent interaction.

However, van der Waals bonds often are important in the packing of amino acids inside a folded protein and in the interactions between adjacent bases stacked within the DNA double helix.

They also can mediate specific interactions because they become collectively strong if the interacting molecules have precisely complementary shapes and can approach one another closely.

VII. Water is a nucleophile.

Water often is a reactant in biochemical reactions.
The unshared pairs of electrons in water molecules can behave as nucleophiles which can attack an electrophilic center in another molecule.
A good example where water serves as a nucleophile is in the hydrolysis of peptide bonds.
Although this is a favorable reaction, peptide bonds are actually quite stable due to the fact that the activation energy for this reaction is quite high.
Thus the reaction is very slow at physiological temperatures and pH unless catalyzed by an enzyme.

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Topic 1: Properties of Water Cont..3
VIII. Ionization of water.

As a prelude to our discussion of pH, we need to discuss the ionization of water, as it is through this reaction that solution pH ultimately is established.

A water molecule has a slight tendency to undergo a dissociation reaction whereby a proton is lost to another water molecule.

The products of this reaction are a hydronium ion (H3O+) and a hydroxyl ion (OH-).

H2O + H2O      H3O+ + OH-

A hydronium ion can donate its proton to another molecule and hence is considered to be an acid (proton donor).

A hydroxyl ion can accept a proton from an acid and thus is called a base (proton acceptor).

The ionization reaction commonly is written as

H2O       H+ + OH-

Water has a finite and defined capacity to ionize, and the ionization process has a characteristic equilibrium constant at a given temperature.

[H+][OH-]

Keq =[H2O]

Keq for water has been experimentally determined by measuring the electrical conductivity of pure water. (Note, electrical conductivity is proportional to the levels of ions in the water).

The value for Keq = 1.8 x 10-16 M.

This Keq value and the value of the concentration of water ([H2O] = 55.5 M) can be substituted into the equilibrium equation to derive another equation which specifies the amounts of [H+] and [OH-] in any water sample or biochemical buffer:

1.8 x 10-16 M =[H+][OH-](55 M)

KW = (55.5 M)(1.8 x 10-16 M) = [H+][OH-] KW = 1 x 10-14 M2 = [H+][OH-]

The constant, KW, is called the ion product of water. The derivation indicates that the product of the [H+] and [OH-] concentrations in any water sample or buffer always will equal 1 x 10-14 M2.

This result leads directly to a definition of a "neutral solution" and to a definition of the pH scale. A neutral solution is defined as one in which [H+] = [OH-].

When these concentrations are equivalent, [H+] = [OH-] = 1 x 10-7 M. Due to the fact that this equilibrium reaction always is obeyed, the addition of a base which consumes protons leads to an excess of hydroxyl ions and a basic solution.

Likewise the addition of an acid which consumes hydroxyl ions leads to an excess of hydronium ions and an acidic solution.

? Properties of Water cont..4

IX. The pH scale. pH is simply a more convenient (if logarithms are convenient!) way of specifying the concentration of H+ ions in solution. pH is defined as pH = -log [H+] = log (1/[H+]) By using logarithms, concentrations in the range of 1 M to 10-14 M H+ are converted to numbers between 0 and 14. For example, the pH of a neutral solution is pH = -log (1 x 10-7) = 7.0

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Topic 1: Properties of Water cont..5
X. Acid dissociation constants of weak acids.

A. Strong and weak acids and bases.

Most reactions are reversible, and equilibrium is achieved when the rate of the forward reaction becomes equal to the rate of the reverse.

As in the water ionization example shown above, the equilibrium constant (Keq) for a general reaction is defined as the ratio of products to reactants at equilibrium.

A + B -> C + D

Keq =[C][D][A][B]

Remember, all reactions have a characteristic Keq at a defined temperature.

Many biomolecules (such as amino acids) are weak acids. Unlike strong acids (HCl, H2SO4, etc.) which completely dissociate when dissolved in water, weak acids only partially dissociate.

Equilibrium reactions for the dissociation of strong acids, strong bases, and the weak acid, acetic acid (CH3COOH) are shown below.

HCl   → H+   + Cl- (100% dissociated)
NaOH   → Na+ + OH-(100% dissociated)
CH3COOH         CH3COO- + H+(<<1% dissociated) (conj. acid)       (conj. base)
In the case of a weak acid, the two species in solution at equilibrium are called the conjugate acid-conjugate base pair.

B. Equilibrium constant (Keq) and the pKa.

The equilibrium constant for dissociation of a weak acid (HA) is

HA              H+     +     A-
(conj. acid)                  (conj. base)

[H+][A-]Keq =[HA]

The equilibrium constant for acid dissociation is more commonly called the acid dissociation constant, Ka, and Ka = Keq. Note, "the higher the Ka, the stronger the acid."

As in the case of pH, biochemists typically use "pKa" values instead of Ka values for weak acids. pKa is defined in the same manner as pH,

pKa = -log Ka = log (1/Ka)

When comparing pKas, "the lower the pKa, the stronger the acid.

C. Henderson-Hasselbalch equation.

The Henderson-Hasselbalch equation describes the quantitative relationship between pH and pKa in buffer solutions. In fact, a titration curve can be plotted using it.

The HH equation will be derived in class starting from the equation specifying the equilibrium constant for ionization of a weak acid, Ka = [H+][A-]/[HA]. The final form of the HH eq is

pH = pKa + log ([A-]/[HA])

or

pH = pKa + log

[conjugate base] [conjugate acid]

The equation indicates that the pH of a solution depends on the pKa and the ratio of conjugate base to conjugate acid components present.

The equation can be used to calculate the pH of a solution of a weak acid when the ratio of [A-]/[HA] is known, or alternatively, to calculate the ratio of [A-]/[HA] when the pH is known.

D. Measurement of pKa by titration.

pKa values are measured experimentally by titration. In a titration experiment, a weak acid in solution is converted to its conjugate base by the addition of a strong base.

Remember that a strong base (e.g., NaOH) will stoichiometrically (1 part-to-1 part) convert a weak acid to its conjugate base form.

Thus, the numbers of moles of weak acid in solution is the same as the number of moles of strong base needed to convert all conjugate acid molecules to their conjugate base form.

A sample equation for titration of acetic acid is shown below.

CH3COOH + Na+ + OH-    →   CH3COO- + Na+ + H2O During the titration, two equilibrium reactions are occurring simultaneously.

1)        H+ + OH-       H2O

and

2)       CH3COOH        H+ + CH3COO-

That is, as an OH- ion is added to the solution, it combines with an H+ ion present in solution.

When the H+ ion is removed, a molecule of the conjugate acid form of acetic acid dissociates a proton to restore equilibrium.

The end result of the titration is that the base converts all of the CH3COOH present to CH3COO-.

Likewise, a strong acid (e.g., HCl) will stoichiometrically convert a weak base to its conjugate acid.

CH3COO- + H+ + Cl-    →   CH3COOH + Cl-

The pKa turns out (as will be proven mathematically using the Henderson-Hasselbalch equation, see below) to be the midpoint of the plot of a titration curve.

Note, that the pH changes rapidly at the ends of the titration curve, and modestly in the middle for a given amount of base added.

The change in pH is least at the midpoint of the curve, i.e., when 0.5 equivalents of strong base have been added.

This middle region of the curve is the optimum buffering region for the weak acid, i.e., the pH changes least on addition of a strong base or strong acid.

At the midpoint [CH3COOH] = [CH3COO-].

Problem 1. Why does pH = pKa at the midpoint of the titration curve?

At the midpoint, [A-] = [HA]. Thus,

pH = pKa + log (1/1)
pH = pKa + 0
pH = pKa

Problem 2. What is the ratio of [A-]/[HA] for a weak acid (pKa = 7.2) at pH = 8.4?

8.4 = 7.2 + log ([A-]/[HA])
1.2 = log ([A-]/[HA])
101.2 = [A-]/[HA]
15.8/1 = [A-]/[HA]

Problem 3. How does pH vary as a function of the ratio of [A-]/[HA]? [A-][HA]                          pH
100/1                          pH = pKa + 2
10/1                            pH = pKa + 1
1/1                              pH = pKa
1/10                            pH = pKa - 1
1/100                          pH = pKa - 2

The titration behavior and its quantitative treatment using the HH equation are similar when dealing with weak acids that have more than one dissociable proton, e.g., phosphoric acid

The only difference is that instead of having one plateau, the titration curve of a "polyprotic" compound shows a plateau for each of its dissociable protons.

The midpoint of each plateau is the pKa for the acid group that is giving up the proton, and one conjugate acid/base pair predominates in solution across each plateau.

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Topic 1: Properties of Water cont..6
XI. Buffers.

A buffer is "a solution that tends to resist a change in pH on addition of a small amount of strong acid or base."

As shown above for the acetic acid titration curve, the pH of a solution undergoing titration changes minimally near the midpoint of the curve.

The optimum buffering power of the solution occurs at the midpoint (where pH = pKa, and [CH3COOH] = [CH3COO-]).

In practice, the optimum buffering region extends about 1 pH unit on either side of the pKa.

In this pH range, buffering power is best because the concentrations of both buffering species, HA and A-, are the highest.

Buffers are selected based on their pKa values and the range of pHs to be buffered. For example, acetic acid (pKa = 4.76) is a good buffer for the range 3.76 < pH < 5.76, whereas the compound "Tris" (pKa ~ 8.0) is a good buffer for the range 7.0 < pH < 9.0.

The main buffering agent inside cells is phosphate. In this case it is the second dissociation reaction with pK2 = 7.2, that provides buffering.

H2PO4-          H+    +   HPO42-

In the blood, the CO2-carbonic acid-bicarbonate system is used for buffering.

Here the major forms responsible for buffering are carbonic acid and bicarbonate. While the pKa for the reaction

H2CO3          H+ + HCO3-

is only 6.4, H2CO3 readily converts to CO2 and H2O which tends to shift the carbonic acid- bicarbonate equilibrium to the left (to a higher pKa) which is closer to the pH of blood. Interestingly, hemoglobin also is a buffering agent in the blood, and the mechanism by which it acts as a buffer will be described later.

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Topic 1: Properties of Water Cont..7
Logarithms and Antilogarithms.

Definition of a logarithm: The logarithm of number is the value of the exponent that is needed to express the number as a power of 10.

Relationship between logarithmic and exponential equations:
A logarithmic equation is just another form of the more familiar exponential equation. That is log10 y = x expresses the same relationship as y = 10x

When you take the logarithm of a number, you are calculating the exponent x. In taking the antilogarithm of a number, you are converting the number back to its exponential form, i.e., the number written in scientific notation.

Relationships between scientific notation and logarithms.

y          y written in scientific notation            x = log10y

0.0000001       1 x 10-7           -7
0.000001         1 x 10-6           -6
0.00001           1 x 10-5           -5
0.0001             1 x 10-4           -4
0.001               1 x 10-3           -3
0.01                 1 x 10-2           -2
0.1                   1 x 10-1           -1
1                      1 x 100            0
10                    1 x 101            1
100                  1 x 102            2
1,000               1 x 103            3
10,000             1 x 104            4
100,000           1 x 105            5
1,000,000        1 x 106            6
10,000,000      1 x 107            7

Application to pH.

By analogy to the above, pH = - log10 [H+] expresses the same relationship as 10-pH = [H+].

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Topic One: Summary
General properties of water

Hydrogen bonding in water

Ionization of water
Henderson-Hasselbalch equetion

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Topic Two: Enzymes
INTRODUCTION TO ENZYMES

•    Enzymes are usually proteins (some RNA)

•    In general, names end with suffix “ase”

•    Enzymes are catalysts

–    increase the rate of a reaction

–    not consumed by the reaction

–    act repeatedly to increase the rate of reactions

–    Enzymes are often very “specific” – promote only 1 particular reaction

–    Reactants also called “substrates” of enzyme

Why Enzymes?

Accelerate and control the rates of vitally important biochemical reactions
Greater reaction specificity
Milder reaction conditions
Capacity for regulation
Enzymes are the agents of metabolic function.
Metabolites have many potential pathways
Enzymes make the desired one most favorable
Enzymes are necessary for life to exist – otherwise reactions would occur too slowly for a metabolizing organis
Enzymes DO NOT change the equilibrium constant of a reaction (accelerates the rates of the forward and reverse reactions equally)
Enzymes DO NOT alter the standard free energy change, (Δ G°) of a reaction

1.         Δ G° = amount of energy consumed or liberated in the reaction

2.         Quantity that determines if a reaction is energetically favorable.

3.         Reaction is thermodynamically favorable or SPONTANEOUS if Δ G° is negative. (Note: Spontaneous does NOT mean instantaneous; energy must be supplied to START a reaction which then proceeds with a release of energy)

4.         i.e. Enzymes DO NOT change thermodynamics (can’t make a reaction spontaneous

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Topic Two: Enzymes Lower activation energy
Enzymes DO decrease the activation energy of a reaction (Δ G°‡)
Activation Energy is the energy required to start a reaction.
Related to rate: Less energy needed to start the reaction, the faster it’ll go
Enzymes DO increase the rate of reactions that are otherwise possible by DECREASING the activation energy of a reaction
Lowering the Ea increases the rate constant, k, and thereby increases the rate of the reaction
Think of activation energy as the BARRIER required to make a product.
Most stable product is the one with the lowest energy.
Most reactions require a “push” to get them started! “Push” is called “energy of activation” for reaction - Also represented by E

Transition State:
a.   Old bonds break and new ones form.
b.   Substance is neither substrate nor product
c.   Unstable short lived species with an equal probability of going forward or backward. d.   Strained intermediate

ENZYMES work by LOWERING the activation energy: a greater fraction of molecules can cross the lower barrier and react to form product – just like ball on waves.

-    Lowers the energy of the transition state and activation energy (both forward and reverse!)
-    Increases the rate – since less energy is needed to start the reaction, the faster it will proceed.
-    Increases the rate of both forward and reverse reactions.
-    Enzyme does NOT change the energy of the reactants (substrates) or products - ΔG° constant
-           Free Energy Value for products is lower that value for reactants (substrates) – Reaction is thermodynamically favorable but needs a push!

How does an enzyme lower the activation energy?
Often by holding reactant molecules in a position where they react more readily.

Shape of enzyme allows reactants to fit into a specific place – called the “active site” of an enzyme Uses energy gained by binding substrate.

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Topic Two: Catalysis
Catalysis

Enzymes poise all the reactants in a arrangement that allows the reaction to proceed more rapidly than it would otherwise.

•    Enzymes physically interact with their substrates to effect catalysis
•   E + S ↔ ES ↔ ES  EP ↔ E + P
where…
E = enzyme
ES = enzyme/substrate complex
ES* = enzyme/transition state complex
EP = enzyme/product complex
P = product

•    Substrates bind to the enzyme’s active site
–    pocket in the enzyme
–          Binding site = where substrate binds; area that holds substrate in place via non- covalent interactions
–    Catalytic site = where reaction takes place

THE PLAYERS:
Binding site
Catalytic site
Enzyme
Substrate

First Step: Enzyme binds to substrate molecule to form an enzyme – substrate complex
E + S ↔ ES

E                     +              S                                                    ES

Second Step: Formation of the transition state complex where the bound substance is neither product nor reactant - ES ↔ ES*
Note Change!! ES ≠ ES*

ES                                                                   ES*

Third Step: Formation of the enzyme – product complex         ES*  EP

ES*                                                                                  EP Fourth Step: Release of product   EP ↔ E + P
EP                                                      E                     +                     P
Once product is released, enzyme is unchanged and can carry out another reaction – only one at a time!
Enzyme Catalysis
Enzymes are proteins that provide a 3D surface for catalysis

Enzymatic cleavage of Sucrose:

Once product is released, enzyme is available to accept another substrate molecule.

Enzyme can only work on one substrate molecule at a time and is NOT changed during the reaction.
            ES/ES*

ENZYMATIC CLEAVAGE OF SUCROSE

FIRST STEP: SUBSTRATE BINDING

BINDING OF A DIPEPTIDE TO A HYPOTHETICAL ACTIVE SITE

Dipeptide cleaved into 2 aa

ACTIVE SITE: Pocket in the enzyme where substrates bind and catalytic reaction occurs

•    Has SPECIFICITY – can discriminate among possible substrate molecules

1.   Others recognize a functional group (Group specificity)

2.   Some only recognize one type of molecule (e.g. D vs L isomers) (Absolute specificity)

•    Active site is a relatively small 3-D region within the enzyme

1.   As we saw, typically a small cleft or crevice on a large protein

•    Substrates bind in active site by weak non-covalent interactions

A. hydrogen bonding

B. hydrophobic interactions

C. ionic interactions

•           The interactions hold the substrate in the proper orientation for most effective catalysis

•   The ENERGY derived from these interactions = “BINDING ENERGY”

•           Using this energy, enzymes both lower the activation energy and stabilize the transition state complex (ES). Each non-covalent interaction provides energy to stabilize the transition state.

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Topic Two: MODELS FOR ENZYME/SUBSTRATE INTERACTIONS
TWO MODELS FOR ENZYME/SUBSTRATE INTERACTIONS:

1.       Lock and Key Model:

A. Substrate (key) fits into a perfectly shaped space in the enzyme (lock)

B. As we’ve said, there is lots of similarity between the shape of the enzyme and the shape of the substrate

C. Highly stereospecific

D. Implies a very RIGID

inflexible active site

E. Site is preformed and rigid

2.       Induced Fit Model         (Hand in Glove analogy)

A. Takes into account the flexibility of proteins

B. A substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S

C. Change in protein configuration leads to a near perfect fit of substrate with enzyme

Induced Fit Model

Glucose binding to hexokinase: Note Conformational Change in Enzyme

D. When substrate is completely held in active site, it takes on characteristics of the

TRANSITION STATE for the reaction

(Non-covalent interactions between the enzyme and substrate change the 3-D structure of the active site, conforming the shape of the active site to the shape of the substrate in its transition state conformation (E + S  ES)

i.   Enzyme puts substrate in correct ORIENTATION to make the reaction proceed

ii.   Enzyme substrate in close PROXIMITY to groups on the enzyme necessary for catalysis—

1.   Catalysis is begun by amino acids making up the active site of the enzyme; R groups are usually crucial in catalysis.

Enzymes Lower the Activation

Energy of the Reaction

How does an enzyme lower Ea?

By Stabilizing the Transition State!

•  Puts molecules in close proximity to react

•    increases the local concentration of reactants)

•  Puts molecules in correct orientation

•    Reactants are not only near each other on enzyme, they're oriented in optimal position to react, making it possible to always collide in the correct orientation.

Proximity & Orientation

iii.   Called “transition state theory”

1.   Enhances the formation of and stabilize the highly energetic transition state

2.   Transition state binds more tightly that substrate or product

•Enzyme binds tightly to the transition state species (i.e. substrates that have been strained toward the structures of the product!

i.   Binding energy helps reach and stabilize the transition state 

lowers activation energy  increases rate!

•Transition state stabilization accomplished through close complementarity in shape and charge between the active site and the transition state.

Reaction proceeds – bonds are broken and new ones formed, transforming S P

Following catalysis, the product(s) no longer fits the active site and is released

A. Many drugs that act as inhibitors of an enzyme are designed to resemble the substrate transition state.

Transition-State Analog Enzyme Model

The binding of the substrate results in the distortion of

the substrate and enzyme in ways that makes the chemical reaction easier.

Enzyme active sites are complementary not to the substrate per se, but to the transition state through which substrates pass as they are converted into products during an enzymatic reaction

Enzymes bind the transition state structure more tightly than the substrate

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Topic Two: MODELS FOR ENZYME/SUBSTRATE INTERACTIONS
TWO MODELS FOR ENZYME/SUBSTRATE INTERACTIONS:

1.       Lock and Key Model:

A. Substrate (key) fits into a perfectly shaped space in the enzyme (lock)

B. As we’ve said, there is lots of similarity between the shape of the enzyme and the shape of the substrate

C. Highly stereospecific

D. Implies a very RIGID

inflexible active site

E. Site is preformed and rigid

2.       Induced Fit Model         (Hand in Glove analogy)

A. Takes into account the flexibility of proteins

B. A substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S

C. Change in protein configuration leads to a near perfect fit of substrate with enzyme

Induced Fit Model

Glucose binding to hexokinase: Note Conformational Change in Enzyme

D. When substrate is completely held in active site, it takes on characteristics of the

TRANSITION STATE for the reaction

(Non-covalent interactions between the enzyme and substrate change the 3-D structure of the active site, conforming the shape of the active site to the shape of the substrate in its transition state conformation (E + S  ES)

i.   Enzyme puts substrate in correct ORIENTATION to make the reaction proceed

ii.   Enzyme substrate in close PROXIMITY to groups on the enzyme necessary for catalysis—

1.   Catalysis is begun by amino acids making up the active site of the enzyme; R groups are usually crucial in catalysis.

Enzymes Lower the Activation

Energy of the Reaction

How does an enzyme lower Ea?

By Stabilizing the Transition State!

•  Puts molecules in close proximity to react

•    increases the local concentration of reactants)

•  Puts molecules in correct orientation

•    Reactants are not only near each other on enzyme, they're oriented in optimal position to react, making it possible to always collide in the correct orientation.

Proximity & Orientation

iii.   Called “transition state theory”

1.   Enhances the formation of and stabilize the highly energetic transition state

2.   Transition state binds more tightly that substrate or product

•Enzyme binds tightly to the transition state species (i.e. substrates that have been strained toward the structures of the product!

i.   Binding energy helps reach and stabilize the transition state 

lowers activation energy  increases rate!

•Transition state stabilization accomplished through close complementarity in shape and charge between the active site and the transition state.

Reaction proceeds – bonds are broken and new ones formed, transforming S P

Following catalysis, the product(s) no longer fits the active site and is released

A. Many drugs that act as inhibitors of an enzyme are designed to resemble the substrate transition state.

Transition-State Analog Enzyme Model

The binding of the substrate results in the distortion of

the substrate and enzyme in ways that makes the chemical reaction easier.

Enzyme active sites are complementary not to the substrate per se, but to the transition state through which substrates pass as they are converted into products during an enzymatic reaction

Enzymes bind the transition state structure more tightly than the substrate

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Topic Two: MODELS FOR ENZYME/SUBSTRATE INTERACTIONS
TWO MODELS FOR ENZYME/SUBSTRATE INTERACTIONS:

1.       Lock and Key Model:

A. Substrate (key) fits into a perfectly shaped space in the enzyme (lock)

B. As we’ve said, there is lots of similarity between the shape of the enzyme and the shape of the substrate

C. Highly stereospecific

D. Implies a very RIGID

inflexible active site

E. Site is preformed and rigid

2.       Induced Fit Model         (Hand in Glove analogy)

A. Takes into account the flexibility of proteins

B. A substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S

C. Change in protein configuration leads to a near perfect fit of substrate with enzyme

Induced Fit Model

Glucose binding to hexokinase: Note Conformational Change in Enzyme

D. When substrate is completely held in active site, it takes on characteristics of the

TRANSITION STATE for the reaction

(Non-covalent interactions between the enzyme and substrate change the 3-D structure of the active site, conforming the shape of the active site to the shape of the substrate in its transition state conformation (E + S  ES)

i.   Enzyme puts substrate in correct ORIENTATION to make the reaction proceed

ii.   Enzyme substrate in close PROXIMITY to groups on the enzyme necessary for catalysis—

1.   Catalysis is begun by amino acids making up the active site of the enzyme; R groups are usually crucial in catalysis.

Enzymes Lower the Activation

Energy of the Reaction

How does an enzyme lower Ea?

By Stabilizing the Transition State!

•  Puts molecules in close proximity to react

•    increases the local concentration of reactants)

•  Puts molecules in correct orientation

•    Reactants are not only near each other on enzyme, they're oriented in optimal position to react, making it possible to always collide in the correct orientation.

Proximity & Orientation

iii.   Called “transition state theory”

1.   Enhances the formation of and stabilize the highly energetic transition state

2.   Transition state binds more tightly that substrate or product

•Enzyme binds tightly to the transition state species (i.e. substrates that have been strained toward the structures of the product!

i.   Binding energy helps reach and stabilize the transition state 

lowers activation energy  increases rate!

•Transition state stabilization accomplished through close complementarity in shape and charge between the active site and the transition state.

Reaction proceeds – bonds are broken and new ones formed, transforming S P

Following catalysis, the product(s) no longer fits the active site and is released

A. Many drugs that act as inhibitors of an enzyme are designed to resemble the substrate transition state.

Transition-State Analog Enzyme Model

The binding of the substrate results in the distortion of

the substrate and enzyme in ways that makes the chemical reaction easier.

Enzyme active sites are complementary not to the substrate per se, but to the transition state through which substrates pass as they are converted into products during an enzymatic reaction

Enzymes bind the transition state structure more tightly than the substrate

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Topic Two: ENZYME INHIBITION
ENZYME INHIBITION
-    INHIBITORS:
•    Interfere with the action of an enzyme
•    Decrease the rates of their catalysis
•           Inhibitors are a great focus of many drug companies – want to develop compounds to prevent/control certain diseases due to an enzymatic activity
1.   e.g. AIDS and HIV protease inhibitors
•    HIV protease essential for processing of proteins in virus
•           Without these proteins, viable viruses cannot be released to cause further infection
-    Inhibitors can be REVERSIBLE or IRREVERSIBLE
•    Irreversible Inhibitors
o Enzyme is COVALENTLY modified after interaction with inhibitor o Derivatized enzyme is NO longer a catalyst – loses enzymatic activity o Original activity cannot be regenerated
o Also called SUICIDE INHIBITORS
        e.g. nerve gas, VX gas
        Aspirin! Acetylates Ser in active site of cyclooxygenase (COX) enzyme
•    Reversible Inhibitors
o Bind to enzyme and are subsequently released
o Leave enzyme in original condition
o Three subclasses:
        Competitive Inhibitors
        Non-competitive Inhibitors
        Uncompetitive Inhibitors
o Can be distinguished by their kinetics of inhibition
How are inhibitors characterized experimentally?

•   First, perform experiment without inhibitor
o Measure velocity at different substrate concentrations, keeping [E] constant. Choose values of [S]
o Get [S] and V values from experiment
o Take reciprocal of [S] and V values (“1 over”)
o Plot on graph 1/[S] (x) vs. 1/V (y) – don’t use Michaelis-Menten – not reliable
•           Second, do the SAME experiment in parallel using a fixed amount of inhibitor and same values for E and S
•   Get V values and plot together with uninhibited reaction
•           Depending on how the graph looks and using the subsequent equation of the line we can: o Determine type of inhibition (competitive vs. non-competitive vs. un-competitive) o Determine Km and Vmax using the NEW line equations
o Determine Ki = inhibition constant – defined as the dissociation constant for the enzyme-inhibitor complex

1.   COMPETITIVE INHIBITORS

•    Shape and structure of inhibitor is very similar to substrate

•    Inhibitor mimic substrate (or transition state) and fits into the active site

•    Physically blocks substrate’s access into the active site

COMPETITIVE INHIBITION

Many pharmaceutical drugs are competitive inhibitors.

Many are transition state analogs: Competitive inhibitors which mimic the transition state of an enzyme catalyzed reaction (e.g. HIV protease inhibitors such a Saquinavir and Viracept).

•    Competitive inhibitors can be identified by the kinetics of their inhibition

•    Lineweaver-Burk plot shows that in the presence of increasing concentrations of inhibitor, I,

o KM INCREASES
o Vmax STAYS THE SAME

o Effects of competition can be overcome by increasing [S]
o KM gets larger, more substrate needed to reach a given rate, can still, however, ultimately achieve Vmax

o Regardless of the concentration of a competitive inhibitor, a sufficiently high substrate concentration will displace the inhibitor from the active site

o The inhibitor and substrate effectively compete with each other for the site

COMPETITIVE INHIBITORS DESIGNED BY TRANSITION STATE ANALOG APPROACHES

NON-COMPETITIVE INHIBITORS

-   Inhibitor binds to a site OTHER than the active site

-   Binding causes a change in the structure of the enzyme so that it cannot catalyze a reaction

Non-competitive inhibitors can also be identified by the kinetics of inhibition

-          In the presence of a non-competitive inhibitor:

o KM STAYS THE SAME
            Binding of substrate has no effect on inhibition; inhibitor can bind to
ES complex

            Not competing for active site

o Vmax DECREASES
        1/Vmax increases; activity goes down

o The effects of non-competitive inhibition cannot be overcome by increasing [S]

MIXED INHIBITION:

Inhibitor affects both substrate binding and Vmax – KM increases and Vmax decreases
Mixed inhibition is when the inhibitor binds to the enzyme at a location distinct from the substrate binding site. The binding of the inhibitor alters the KM and Vmax.

Similar to noncompetitive inhibition except that binding of the substrate or the inhibitor affect the enzyme’s binding affinity for the other.

Binding affinity for the substrate is decreased when the inhibitor is present.

UN-COMPETITIVE INHIBITORS

-   Inhibitor binds to a site other than the active site, but only when substrate is bound
(Binds to ES complex)

•     Distorts active site; prevents reaction from occurring

•   Un-competitive inhibitors can also be identified by the kinetics of inhibition

  In the presence of an UN-COMPETITIVE inhibitor:

            KM DECREASES (Effectively increases the affinity for the substrate)

•    Vmax DECREASES (Overall rate goes down)
•   The effects of un-competitive inhibition cannot be overcome by increasing [S]

Irreversible inhibitors
! Inhibitors covalently modify the active site – permanent inhibition. Must wait for more enzyme to be made.    Example: Aspirin, nerve gas

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Topic Two: COENZYMES:
COENZYMES:

- Some enzymes require an additional component for activity
-    These      components      are      called
COENZYMES
•           Used at the active site of the enzyme
•           Not   covalently   bound   to   the enzyme
•           Can be small organic molecules or metal ions
•           Many are structurally related to vitamins
•           They are regenerated for further reactions

PROSTHETIC GROUPS are COENZYMES that ARE covalently bound to an enzyme and therefore are always present

REGULATION OF ENZYME ACTIVITY:
-    Regulation means to make an enzyme more or less active
-           In general, regulation is necessary to control the rates of reactions and to properly synchronize all of the metabolic reactions in the cell. Keep it running like a finely tuned machine.
How can you change enzyme behavior in a cell?
Feedback inhibition of metabolic pathways
High concentrations of a downstream product of a pathway signals an upstream enzyme to shut down.
- Induction/repression
•    Change the rate of enzyme synthesis and/or degradation of the enzyme
•    Change cellular distribution of the enzyme

-    Modify the intrinsic properties of the enzyme
•   Non-covalent interactions
1.   Bind regulatory molecules reversibly (e.g. proteins, lipids, small molecules)

• Reversible Covalent Modifications
1. Phosphorylation of serine, threonine or tyrosine
2. Methylation of glutamate residues
o Used in bacteria as food sensor
Regulation by covalent modification of an enzyme

3. Creation or reduction of disulfide bonds
• Irreversible Covalent Modifications
1. Isoprenylation, acylation, palmitoylation – addition of fatty acids and fatty acid derivatives
2. Glycosylation – addition of sugars to Asparagine
3. Proteolytic cleavage
o ZYMOGENS – inactive precursor to an enzyme; activated by cleavage of a specific peptide bond
o Why would this be useful? Let’s look at examples:
          Proteolytic enzymes TRYPSIN and CHYMOTRYPSIN
                  Initially synthesized as trypsinogen and chymotrypsinogen which are both inactive
          Formed in the pancreas where they would do damage if active

                  In the small intestine, where their digestive properties are needed, they are ACTIVATED by cleavage of specific peptide bonds.

ZYMOGENS ARE INACTIVE UNTIL REACH PROPER ENVIRONMENT!!

CHYMOTRYPSINOGEN:

INACTIVE PRECURSOR OF CHYMOTRYPSIN

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Topic 3: AMINO ACIDS
-    All 20 amino acids in pure form are white, crystalline, high-melting solids

-           Amino acids act as: enzymes (catalysts), metabolic intermediates, carriers of energy and waste products and hormones.
-    Amino acids are the building blocks of proteins
-           Proteins are the most abundant macromolecules in living cells. May be 0.1 million different proteins in humans. Play pivotal role in almost every biological process.
-    Generally, proteins composed of the 20 naturally occurring amino acids
-    Only one way to link amino acids together – peptide bond
-    Protein structure and function defined by sequence and type of the amino acids
-    Proteins display great diversity in function and structure

DEFINITION:
Any organic molecule with at least one CARBOXYL group (organic acid) and at least one
AMINO group (organic base)

GENERAL STRUCTURE OF THE 20 AMINO ACIDS:

At physiological pH (~7.4), amino acids exist as zwitterions – positive and negative charge on the same molecule. Dependent upon the pKa of the group. For example:
§ Carboxylic acid functional group
§ Amine functional group
R = biochemical shorthand for “side chain” Protonation of COOH and H2N depends on pH of the solution

General anatomy of an amino acid.
Except for proline and its derivatives, all of the amino acids commonly found in proteins

Have both ACID and BASE groups in same molecule

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Topic 3: STEREOCHEMISTRY OF AMINO ACIDS:

-    Stereochemistry imparts certain characteristics to a compound.
o Every compound has a mirror image
o Sometimes mirror images of a molecule are superimposable in space with the original object and
some are not
o NON-superimposable mirror images = CHIRAL
        Have no plane of symmetry
        General rule: Carbon atom with 4 DIFFERENT atoms or groups bonded to it is chiral.
        Other Chiral examples:
•   Hand or foot
•           Right hand is a mirror image of your left hand – note how they cannot be superimposed
o Superimposable mirror images = ACHIRAL
        Achiral examples:
•   Two plain coffee mugs
•           You can turn the mirror image of the plain mug in space to make it superimposable.

-    Each amino acid (like your hand) also has a mirror image.

These images are NOT superimposable; No matter how you turn the mirror image in space you won’t regain the original. Note no plane of symmetry

These are called ENANTIOMERS = non-superimposable mirror images
-    Most amino acids (except glycine) have four different groups attached to the a-carbon and therefore are chiral or have a chiral center.
-    For glycine, R is hydrogen. Therefore, the mirror images ARE superimposable and NOT chiral. A plane of symmetry exists.
-    Each amino acid except glycine has 2 ENANTIOMERS
-           The enantiomers are classified based on the ability to rotate polarized light – optically active.
o Rotate light in either (+) or (-) direction
o Called D or L enantiomers (again non- superimposable mirror images)
o Both D and L amino acids exist in nature but only L amino acids are used as building blocks for proteins.
o D-amino acids are found in a few rare bacteria in the cell walls.

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Topic 3: CLASSIFICATION AND CHEMICAL CHARACTERISTICS OF EACH AMINO ACID:

-    The “R” group side chains on amino acids are VERY important.
Determine the properties of the amino acid itself
Determine the properties of the proteins that contain those amino acids; Dictate what a protein can and cannot do and how it folds.
Important to remember that biomolecules have three dimensions. It’s this feature that dictates which reactions can take place in a cell.
-    All 20 amino acids have both three letter and one letter abbreviations
-    For example:
Alanine (Ala, A) Cysteine (Cys, C)

-    You are responsible for knowing the three letter and one letter codes and well as the structures of all 20 amino acids.
-    Groups classified by different properties I:    Non-polar side chains (hydrophobic) o II: Polar, uncharged side chains
III: Charged Side Chains
Acidic side chains
Basic side chains

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Topic 3: NON-POLAR (HYDROPHOBIC) SIDE CHAINS
GROUP I: NON-POLAR (HYDROPHOBIC) SIDE CHAINS

- Side chains of Group I aa’s are mainly hydrocarbons – very unreactive amino acids
- 2 subgroups: Aliphatic hydrocarbons & Aromatic hydrocarbons (have benzene rings)
- These amino acids will tend to be buried (away from water) in 3-D structure of proteins
- Non-polar character

1)        ALIPHATIC HYDROCARBONS
a.   Glycine (G, Gly)
i.            R group is hydrogen
ii.            Found in flexible parts of proteins
iii.            Not chiral
iv.            Can be modified by addition of a fatty acid (myristate – 14 Carbon)

b. Alanine (A, Ala)
i.            The model amino acid
ii.            R group is –CH3 (methyl group)

c.   Valine (V, Val) Leucine (L, Leu) Isoleucine (I, Ile)
• Extended aliphatic chains
• Can be branched

d. Methionine (M, Met)
i.   Contains sulfur
ii.   Can interact and bind with metal ions iii.   Often found in metalloproteins

e.   Proline (P, Pro)
i.   Only imino acid
ii.   Affects protein folding
iii.   Often found at bends in protein 3-D structures
iv. Hydroxylation of proline important for the structure of collagen
2) AROMATIC HYDROCARBONS

a.   Phenylalanine (F, Phe) and Tryptophan (W, Trp)
i.   Fluorescent
ii.   UV absorbing at 250-300 nm – can be useful to identify proteins in a
mixture
iii.   Tryptophan is converted to serotonin (5-hydroxytryptamine)
• Serotonin has a sedative effect – gives a pleasant feeling
• Very low levels of serotonin associated with depression
• Extremely high levels produce a manic state

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Topic 3: NEUTRAL (UNCHARGED) POLAR SIDE CHAINS
GROUP II: NEUTRAL (UNCHARGED) POLAR SIDE CHAINS
-    Polar Residues are both buried as well as on the surface of proteins. They either form hydrogen bonds with other polar residues
in the protein or with water. For example, the OH group of Serine can
both donate as well as accept a hydrogen bond:

Serine

a.   Serine (S, Ser) and Threonine (T, Thr)

i.                Polarity contributed by the hydroxyl group (-OH)
ii.                Sugars attach to Ser and Thr to form glycoproteins
iii.        Ser & Thr can have phosphates attached – regulates the activity of some proteins

b. Tyrosine (Y, Tyr)

i.                Fluorescent – absorbs UV light at 280nm (easy to identify)
ii.                Can be phosphorylated (have phosphates attached)
iii.                Derived from phenylalanine
iv.                Converted to catecholamines – includes epinephrine (adrenaline)
• “fight or flight” hormone
• causes release of glucose and other nutrients into the blood and stimulates brain function

•   Tyrosine, Serine, and Threonine
- Can be phosphorylated on hydroxyl groups
- Phosphoserine, phosphotyrosine, phosphothreonine
- Involved in signal transduction pathways

c.   Asparagine (N, Asn) and Glutamine (Q, Gln)

i.                Classified as amides
ii.                Neither acidic or basic iii.                Forms H-bonds
iv.                Asn can be modified with sugars to form glycoproteins

d. Cysteine (C, Cys)
Sulfhydryl side chain (-SH) (gives the polarity)
Cys can be modified by addition of farnesyl or geranylgeranyl
                        groups
            1.         Example – Required for membrane association of Ras proteins

Can oxidize to form disulfide bonds that strengthen protein structure
Disulfide bonds are covalent but reversible upon reduction

Protein------CH2-SH
+
HS-CH2------Protein          Protein------CH2-S-S-CH2------Protein

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Topic 3: Charged Amino Acids
GROUP III: Charged Amino Acids

A. ACIDIC SIDE CHAINS
a.   Glutamate or glutamic acid (E, Glu) and Aspartate or aspartic acid (D, Asp)
Always negatively charged at physiological pH (~7.4)
--------CH2-COO-
B. BASIC SIDE CHAINS
All gain a proton at physiological pH (Positively charged)

a.   Lysine (K, Lys)
b. Arginine (R, Arg)
c.   Histidine (H, His)

His is ionizable near physiological pH; therefore can act as a proton donor OR acceptor depending on the pH in the local environment. ~50% protonated under physiological conditions

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Topic 3: General Rules
SUMMARY & SOME GENERAL RULES:

• Charged, hydrophilic residues are hardly ever buried – tend to cluster on outside of a protein in water

• Polar residues are usually found on the surface of the protein, but can be buried.

• The inside, or core of a protein contains mostly non-polar, hydrophobic amino acids.

• Non-polar residues are also found on the outside of proteins.

• Recognition of one biological molecule by another (i.e. surface-surface contacts) can utilize charged, polar and non-polar interactions.

OTHER AMINO ACIDS:

•           Some other amino acids are derivatized once incorporated into proteins

•           Hydroxyproline and hydroxylysine

o Found in collagen, the principle component of connective tissue

o Proline and lysine modified after incorporation

o Modifications essential for maintaining normal

connective tissues in tendons, cartilage, bones, teeth, skin

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Topic 3: Amino Acids as Acids, Bases and Buffers:
Amino Acids as Acids, Bases and Buffers:

-    Amino acids are weak acids
-           All have at least 2 titratable protons, and therefore have 2 pKa’s
o α-carboxyl (-COOH)
o α-amino (-NH3+)
-           Some amino acids have a third titratable proton in the R group and therefore a third pKa

AMINO ACIDS AS WEAK ACIDS:

- Properties of amino acids in proteins and peptides are determined by the R group but also by the charges of the titratable group. Will ultimately affect protein structure.
-           Important to know which groups on peptides and proteins will be protonated at a certain pH.
The degree of dissociation depends on the pH of the solution.

The first dissociation is the carboxylic acid group (using glycine as an example):

+NH3CH2COOH !" +NH3CH2COO- + H+[+NH3CH2COO-][H+]

The second dissociation is the amino group in the case of glycine:+NH3CH2COO-   !" NH2CH2COO-   + H+[NH CH COO-][H+]

Ka1 = ---------------------------[+NH3CH2COOH]

Ka2 = ---------------------------[+NH CH COO-]

How do we do this?? Example – Alanine

1.       Draw the fully protonated structure

Q: Which protons come off when?
A: Look at pKa table for amino acids
Alanine has 2 pKas:
α-COOH (pKa = 2.3) comes off first (has lower pKa)
α-NH3+ (pKa = 9.9)
Others come off SEQUENTIALLY in ascending order of pKa.
2.         Write out structures for sequential deprotonation and place pKa values over the equilibrium arrows.

Alanine
Fully protonated         1st proton removed     2nd proton removed
Net charge = +1          Net charge = 0            Net charge = -1
So, from looking at the net charges, at different pH’s, amino acids can have different charges! Very important for protein structure!!
Remember that the pKa = pH when ½ of an available amount of an ionizable group is ionized.
-    Let’s take a look at the titration
curve for Alanine
-    Looks very much like what we
saw for acetic acid last time except that it has 2 midpoints (pKa’s) – one for each proton α-COOH and α-NH3+
-    At beginning, all protonated
-Need one equivalent of base for each proton
-    At each HALF equivalent = pKa
o 50% protonated/50%deprotonated
-    At end all deprotonated
-For our purposes, to determine whether the proton is ON or OFF at a certain pH use the following RULES
-
o pH = pKa        Equal amounts of protonated and deprotonated species exist
if pH is LESS than the pKa of a particular group, that group will be predominantly protonated

if pH is GREATER than the pKa of a particular ionizable group, that group will be predominantly deprotonated

For example: Alanine at different pH’

At pH 1.5:       pH is less than the pKa of both the α-COOH
and the α-NH3+, therefore, both protons are ON
At pH 7: pH is greater than the pKa of the α-COOH  H+ OFF
pH is less than the pKa of the α-NH3+  H+ ON

At pH 10.5      pH is greater than the pKa of the α-COOH  H+ OFF
pH is greater than the pKa of the α-COOH  H+ OFF
Amino acids can be separated on the basis of their charges at a certain pH

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Topic 3: HOW ARE AMINO ACIDS MADE?

-           Many organisms can make all 20 of the amino acids
o Bacteria, yeast, and plants
-           Some amino acids are made from common metabolic intermediates directly
o For example, alanine is made from pyruvate (transamination of pyruvate with glutamate as the amino donor)
-           Some amino acids are made as products from long and complex pathways
o For example, aromatic amino acids are made from the shikimic acid pathway
-           Humans and other animals CANNOT make some of the 20 amino acids
o These are ESSENTIAL AMINO ACIDS

Arginine and Histidine are essential only in babies or in people with extreme metabolic stress disease – Conditional Essential Amino Acids
-    Foods vary in “protein quality”
o Content of essential amino acids

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Topic 3: PEPTIDES and PROTEINS
-    Learned basic chemistry of amino acids – structure and charges
-           Chemical nature/charges of amino acids is CRUCIAL to the structure and function of proteins
-    Amino acids can assemble into chains (peptides, polypeptides, proteins)
o Can be very short to very long
Dipeptide = two amino acids linked
Tripeptide = three amino acids linked
-    Amino acids sometimes called RESIDUES
-    Identity and function of a protein or peptide is determined by
o Amino acid composition
o Order of amino acids in the chain
o Enormous variety of possible sequences
e.g., if you have a protein with 100 aa, there are 1.27 X 10130 possible sequences!
-    Amino acids are linked by COVALENT BONDS = PEPTIDE BONDS
-    Peptide bond is an amide linkage formed by a condensation reaction (loss of water)
-    Brings together the alpha-carboxyl of one amino acid with the alpha-amino of another
-    Portion of the AA left in the peptide is termed the amino acid RESIDUE
o Amino acids sometimes called RESIDUES
-    R groups remain UNCHANGED – remain active
-    N-terminal amino and C-terminal carboxyl are also available for further reaction
-    Reaction is NOT thermodynamically favorable (not spontaneous)
o Need energy and other components and instructions to correctly assemble
This is the process of protein translation

FORMATION OF THE PEPTIDE BOND

-    Peptides are always written in the N-C direction
-           Each peptide has ONLY ONE free amino group and ONE free carboxyl group; others are neutralized by formation of the peptide bond
-

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Topic 3: IMPORTANT FEATURES OF THE PEPTIDE BOND:

1.     Peptide bonds have double bond character resulting from resonance stabilization

(C-N bond has 40% double bond character) C-N and C-O have partial double bond character

Resonance Results in Partial Double Bond Character of the Peptide Bond (Amide Bond):

Rotation Restricted

-    Results in the peptide bond being PLANAR

o C, N, H, O are all in the same plane

(Cα’s are also in plane)

o p orbitals can overlap to form partial double bonds between the nitrogen and carbon and the carbon and oxygen

-           Stronger than a normal bond because of the double bond character

-    No tendency to protonate

-    No significant rotation occurs around the peptide bond itself.

-           PEPTIDE BACKBONE is made up of these rigid planar units with limited rotation around the Cα carbons – N- Cα1 (phi) and the Cα2−C (psi) bonds. These bonds link the peptide groups in the peptide chain

o Crucial for protein structure

o Rotation can occur at the

α−carbons

o However, rotation is limited due to steric hindrance (R groups)

o These limitations limit the shapes that can be assumed by protein chains

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Topic 3: Summary
This topic discusses;

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Topic 3: ELECTROLYTES
Electrolytes are body substances which dissociate in solution.

These includes; NaCl, & KCl, which dissociate in solution into sodium (Na+), potassium (K+) and chloride (Cl–) ions.

Water molecules completely surround these dissociated ions and prevent union of +vely charged particles with –vely charged ones.

The +ve ions are called cations and negatively (–vely) charged ions are called anions.

Law of electrical neutrality: Fluid in any body compartment will contain equal number of +vely charged and –vely charged ions.

Solutes in body fluids:

Solutes in body fluids are mainly of three categories.

1. “Organic” compounds of small molecular size like glucose, urea, uric acid, etc.
They are “nonelectrolytes” as they do not dissociate or ionise in solution.
Since these substances diffuse relatively freely across cell membrane they are not important in the distribution of water.
If it is present in large quantities, however, they aid in retaining water and thus do influence total body water.

2. “Organic” substances of large molecular size, mainly the proteins. Effect of protein fractions of the plasma and tissues is mainly on the transfer of fluid from one compartment to another and not on the total body water.

3. Inorganic “electrolytes”: Because of the relatively large quantities of these materials in the body, they are the most important both in distribution and retention of body water.
Electrolytes composition of ECF: Both plasma (IVF) and tissue fluid (ITF) may be considered as one single compartment for all practical purposes as both resemble each other and both differ grossly from ICF.
Electrolytes composition of TF is similar to plasma except that Cl– largely ‘replaces’ proteins as anion.

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Topic 3: Electrolytes of plasma and tissue fluid Cations & Anions in mEq/L
(a)    Plasma Na+ = 143 Cl– = 103 K+ = 5 HCO– 3 = 27 Ca++ = 5 HPO= 4 =2 Mg++ = 2 SO = 4 =1 Total = 155 Proteins– = 16 Organic acids– =6 Total = 155
(b)   Tissue fluid Na+ = 145 Cl– = 116 K+ = 5 HCO– 3 = 27 Ca++ = 3 HPO= 4 =3 Mg++ = 2 SO = 4 =2 Total = 155 Proteins– =1 Organic acids– =6 Total = 155 Electrolytes composition of ICF: ICF contains 195 mEq of cations and anions. Values of different electrolytes in

Electrolytes composition of ICF: ICF contains 195 mEq of cations and anions. Values of different electrolyte
ICF differs in different tissues. But chief cations are K+ and then Mg++.

These are balanced by the chief anions PO= 4 and next by Pr–. About two-thirds of K+ within cells is “protein-bound”, while remaining one-third is ‘free’ which exchanges with ECF. Cl–, HCO– 3 and Na+ are present in intracellular fluid only in minimal amounts.

Total electrolytes concentration is higher than that of the ECF. The phosphates of the cells are phosphoric esters of hexoses, creatine phosphate, ATP and inorganic phosphates.

Electrolytes of ICF
Cations mEq/L Anions mEq/L K+ = 150 HPO= 4 = 110 Mg++ = 40 Protein– = 50 Na+ = 5 SO = 4 = 20 Total = 195 HCO– 3 = 10 Cl– =5 Total = 195

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Topic 3: Normal Fluid and Electrolytes Exchanges in the Body
Under normal conditions in health, the relative volumes of water in above three compartments are kept constant.

Water can pass freely through the membrane which divides plasma from tissue fluid; and tissue fluid from intracellular fluid; but distribution of water is controlled by the osmotic pressure exerted by substances present in each compartment, i.e. the electrolytes and protein molecules.

• Membrane separating ICF from tissue fluid is Semipermeable called as slow membrane by Darrow, and it allows only passage of water but not electrolytes and protein molecules in health.

Normally, there is osmotic equilibrium between these two compartments, but if this is disturbed, water is drawn from the compartment with lower osmotic pressure into that with higher osmotic pressure until equilibrium is restored.

The osmotic imbalance between these two compartments results in water being either sucked out of the cells (producing cellular dehydration) or water is drawn into the cells (producing cellular oedema) to restore the balance.

• Membrane separating the vascular compartment from tissue fluid is more permeable, called as Rapid membrane by Darrow, to water and electrolytes but not to protein molecules.

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Topic 3: Electrolytes Movement In and Out of Cells

• Much higher concentration of Na+ and Cl– in interstitial fluid and K+ in intracellular fluid are accompanied by a difference in electrical potential.
The resting skeletal muscle cells being about 90 mv –ve to the interstitial fluid. It is believed that the Lipid-protein membrane plays an important role in determining and maintaining these differences in concentration and potential.

·         K + ions tend to diffuse out of and the Cl– ions into the cells because of their concentration gradients, but this is almost exactly counter-balanced by a tendency to diffuse in the opposite direction due to the difference in electrical potential, i.e. the relative negativity on the inside of the cells tend to keep Cl–out and K+ in.

·         In the case of Na+, however, diffusion into the cells is favoured by both the concentration gradient and electrical potential.
Cells do not allow accumulation of Na+, hence under normal healthy conditions; there must be some mechanism for removing Na+ from the cell, virtually as rapidly as it enters.
Since this has to be accomplished in opposition to forces of concentration and electrical potential, it involves expenditure of energy, derived from cellular metabolism.
This process of “Active transport” of Na+ out of cells (Pumping out) is done by the Sodium Pump, which effectively extrudes Na+ from the intracellular fluid.
This extrusion of Na+ from the cell is associated with splitting of ATP by “Na+ – K+ ATPase” located at the inner surface of the cell membrane. The enzyme is activated by Mg++, has a molecular weight of 2, 50,000 to 3, 00,000.

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Topic 3: Summary
The topics discusses common body electrolytes in terms of

1. Solutes in body fluids

2. Electrolytes of plasma and body fluids

3. Electrolytes of ICF
4. Electrolytes movement in and out of the cell

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Topic 4: VITAMINS

Vitamins are organic compounds occurring in natural foods as utilisable precursors, which are required in minute amounts for normal growth, maintenance and reproduction.
1.      They differ from other organic food stuffs in that:
• They do not enter into tissue structures, unlike proteins.
• Do not undergo degradation for providing energy unlike carbohydrates and lipids.
• Several B complex vitamins play an important role as coenzymes in several energy transformation reactions in the body.
2.      They differ from hormones:
In that, they are not produced within the organism, and most of them have to be provided in the diet.

Classification
All vitamins are broadly divided into two groups according to solubility.
1. Fat-soluble Vitamins
·         Vitamin A
·         Vitamin D
·         Vitamin E, and
·         Vitamin K.

2. Water-soluble Vitamins
(a) Vitamin C (ascorbic acid),
(b)Vitamin B complex group includes:
·         Vitamin B1 (thiamine
·         Vitamin B2 (riboflavin)
·         Niacin (nicotinic acid)
·         Vitamin B6 (pyridoxine)
·         Pantothenic acid
·         αα αα α-Lipoic acid
·         Biotin
·         Folic acid group
·         Vitamin B12 (cyanocobalamine).

Other water-soluble vitamins included in this group are: • Inositol
·         Para-amino benzoic acid (PABA)
·         Choline.

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Topic 4: FAT SOLUBLE VITAMINS
There are three sub-unts of vitamin A;

Retinol- Vit. A alcohol
Retinal – Vit. A aldehyde
Retinoic acid –

Dietary Sources
• Animal sources: Liver oil, butter, milk, cheese, eggyolk.
• Plant sources:
In the form of provitamin carotene, tomatoes, carrots, green-yellow vegetables, spinach, and fruits such as mangoes, papayas, corn, sweet potatoes. Spirulina species (algae) have been found to be a good source of vitamin A.ViT. A acid

Functions of Vitamin A
Pay role in vision. i.e. rods and cones in the retina are made of vit. A. its deficiency leads to night blindness.
Play role in testis formation and sperm maturation. Its deficiency leads to infertility.
Play role in epithelisation of the skin. Its deficiency leads to dry scaly skin.
Play role in bone and teeth development, its deficiency leads to cancellous bones and thin teeths.
Are involved in growth, i.e play role in cell division and defferenciation. Its deficiency leads to retarded growth.
Play role in protein and DNA metabolism

Deficiencies cause:

• Night blindness, xerophthalmia (keratin deposits in cornea), macular degeneration.
• Skin and mucous membrane dryness and infection, keratin deposits.
• Anemia
• Developmental defects – bones, teeth, immune system, vision

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Topic 4: FAT SOLUBLE VITAMINS Cont..2
VITAMIN D
Vitamin D3 or cholecalciferol occurs in fish liver and also produced human skin by ultraviolet light.
The inactive natural precursors of the vitamin D are the ‘provitamins’
• Ergosterol: Provitamin D2 found in plants and
• 7-dehydrocholesterol: Provitamin D3 found in the skin.
Transformation from inactive provitamin to the active vitamin is accomplished by the ultraviolet rays

Dietary Sources
Fish liver oil is the richest source of vitamin D. other sources are, Egg-
yolk, margarine, butter, cheese, etc. Ergosterol is widely distributed in
plants.
Bile salts help in absorption of vitamin D from duodenum and jejunum.
After absorption, it is carried in chylomicron droplets of the lymph in
combination with serum globulin in blood plasma.
The biologically active form of vitamin D calcitriol, amd it is
synthesised in liver and kidneys.

(a)   Synthesis of 25-OH-D3 in Liver (Calcidiol)
• Vitamin D2 and/or D3 binds to specific D binding protein and is transported to liver.
• It undergoes hydroxylation at 25 position, by the enzyme 25-hydroxylase, in the endoplasmic reticulum of the mitochondria of liver cells.
• Coenzyme/cofactors required are: • Mg ++ • NADPH, and • Molecular O2

(b) Synthesis of 1, 25-di –OH-D3 (Calcitriol) in Kidneys
• 25-OH-D3 is bound to a specific vitamin D binding protein and is carried to kidneys.
• It undergoes hydroxylation at 1-position, by the enzyme 1 αα αα α -hydroxylase, in the endoplasmic reticulum of mitochondria of proximal convoluted tubules of kidney.
• The reaction is a complex three component monooxygenase reaction requiring Mg++, NADPH and molecular O2 as coenzymes/cofactors.
• In addition, at least three more enzymes are required. They are:
• Ferrodoxin reductase
• Ferrodoxin, and
• Cytochrome P450 This system produces 1,25-di-OH-D3 (calcitriol) which is the most potent metabolite of vitamin D

FUNCTIONS OF VITAMIN D
• Intestinal absorption of calcium and phosphate:
• Mineralisation of bones: Mineralisation of bones is promoted by 1, 25, (OH)2D3 as well as 24, 25(OH)2D3.
• Renal reabsorption of calcium and phosphorus is also done by 1,25(OH)2D3 in similar way.
• It lowers the pH in certain parts of the gut such as colon and produces increase in urinary pH.
• It counteracts the inhibitory effect of calcium ions on the hydrolysis of phytate.
• In physiologically compatible intake it is found to increase the citrate content of bone, blood, tissues and urinary level.

Deficiency of Vitamin D—Clinical Aspect
1.      Produces rickets in growing children and osteomalacia in adults.
2.      Osteomalacia The deficiency of vitamin D in adults is osteomalacia which is rare
3.      Renal Osteodystrophy When renal parenchyma is lost or diseased quite significantly, it is unable to form calcitriol and calcium absorption is impaired

HYPERVITAMINOSIS D
1. Immediate effects: Include anorexia, thirst, lassitude, constipation and polyuria. Followed later on by nausea, vomiting and diarrhoea.
2. Delayed effects: Persistent hypercalcaemia and hyperphosphataemia may produce:

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Topic 4: FAT SOLUBLE VITAMINS Cont..3
VITAMIN E

Also called Tocopherol

Dietary Sources

Cottonseed oil, corn oil, sunflower oil, wheat germ oil and margarine are the richest sources of vitamin E. It is also found in fair quantities in dry soyabeans, cabbage, yeast, lettuce, apple seeds, and peanuts.

FUNCTIONS OF VITAMIN E

1. Antioxidant Property

This is the most important functional aspect of vitamin E.

• Removal of free radicals: Vitamin E is involved in removal of free radicals and prevents their peroxidative effects on unsaturated lipids of membranes and thus helps maintain the integrity of cell membrane

2. Role in Reproduction,

Vitamin E helps in maintaining seminiferous epithelium intact. However, its deficiency leads to irreversible degenerative changes leading to permanent sterility. Motility of sperms is lost and spermatogenesis is impaired.

. 3. Other Functions

• Tocopherol derivative tocopheranolactone may be involved in synthesis of coenzyme Q or ubiquinone.

• Vitamin E may have some role in nucleic acid synthesis.

Deficiency of Vitamin E

• Muscular dystrophy: Vitamin E deficiency leads to the increased oxidation of polyunsaturated fatty acids in the muscle with a consequent rise in O2 consumption and peroxide production; peroxides may then cause an increase in intracellular hydrolase activity by affecting the lysosomal membranes.

• Hemolytic anemia: Low tocopherol diet may produce low plasma tocopherol, increased susceptibility to hemolysis due to peroxides an

dialuric acid.

Clinical and therapeutic uses:

Vitamin E has been used in;

• Nocturnal muscle cramps (NMC)

• Intermittent claudication (IC)

• Fibrocystic breast disease (FBD)

• Atherosclerosis

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Topic 4: FAT SOLUBLE VITAMINS Cont..4
VITAMIN K:

All vitamin K forms are naphthoquinone derivatives. Vitamins K1 and K2 are the two naturally occurring forms of vitamin K that have been identified.

Dietary sources:

Both vitamin K1 and K2 are mainly found in plants and synthesized by bacteria respectively. Vitamin K1 is present chiefly in green leafy vegetables, such as alfalfa, spinach, cauliflower, cabbage, soybeans, and tomatoes. Vitamin K2 also called Menaquinones is a product of metabolism of most bacteria including the normal intestinal bacteria of higher animal species. Menaquinones (K2) are absorbed from gut to some extent

FUNCTIONS OF VITAMIN K

Blood Coagulation

The main function of vitamin K is the promotion of blood coagulation by helping in the posttranscriptional modifications of blood factors such as prothrombin, and factors II, VII, IX, X.

Calcium Binding Proteins

Vitamin K similarly is found to carboxylate specific glutamate residues of calcium binding proteins of bones, spleen, placenta and kidneys. This enhances the capacity of these proteins to deposit calcium in the tissues concerned.

Role in Oxidative Phosphorylation

Vitamin K is a necessary cofactor in oxidative phosphorylation being associated with mitochondrial lipids

DEFICIENCY OF VITAMIN K

Deficiency of vitamin K is very rare, since most common foods contain this vitamin. In addition, intestinal flora of microorganisms also synthesise vitamin K. However, a deficiency may occur as a result of:

• Prolonged use of antibiotics and sulfa drugs:

• Malabsorption and biliary tract obstruction

. • Spoilt Sweet-clover hay: When consumed by cattle, causes a

                 disease.

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Topic 4: WATER-SOLUBLE VITAMINS

VITAMIN C (ASCORBIC ACID) Synonyms: Antiscorbutic vitamin

• Ascorbic acid is an enediol-lactone of an acid with a configuration similar to that of the sugar L-glucose. • It is a comparatively strong acid, stronger than acetic acid, owing to dissociation of enolic H at C2 and C3.

Dietary sources:

Good sources are citrous fruits—orange/lemon/lime, etc; other fruits like papaya, pineapple, banana, strawberry. Amongst vegetables—leafy vegetables like cabbage and cauliflower, germinating seeds, Green peas and beans, potatoes, and tomatoes. Amla is the richest source.

Considerable amount of vitamin C activity is lost during cooking, processing and storage, because of its water-solubility and its irreversible oxidative degradation to inactive compounds.

METABOLIC ROLE AND FUNCTIONS

1.      Role in Cellular Oxidation-Reduction

The fact that vitamin C is very sensitive to reversible oxidation, Ascorbic acid → ← Dehydroascorbic acid, suggests that it may be involved in cellular oxidationreduction reactions, perhaps serving as hydrogen transport agent

.

2.      Role in Collagen Synthesis

Hydroxyproline and hydroxylysine are important constituents of mature collagen fibres. Precollagen molecules contain the amino acids proline and lysine. They are hydroxylated by corresponding hydroxylases in presence of vitamin C, Fe++ and molecular O2.

3.      Functional Activity of Fibroblasts/Osteoblasts

Ascorbic acid is required for functional activities of fibroblasts, and osteoblasts, and consequently for formation of MPS of connective tissues, osteoid tissues, dentine and intercellular cement substance of capillaries.

4.      Role in Tryptophan

Metabolism Vitamin C is required as a cofactor for hydroxylation of tryptophan to form 5–OH derivative, in the pathway of biosynthesis of serotonin (5–HT).

5.      Role in Tyrosine Metabolism

Required as a cofactor with the enzyme p-OH phenyl pyruvate hydroxylase, which is necessary for hydroxylation and conversion of p-OH phenyl pyruvate to Homogentisic acid.



DEFICIENCY MANIFESTATIONS: SCURVY

The main defect is a failure to deposit intercellular cement substance.

• Capillaries are fragile and there is tendency to haemorrhages.

• Wound healing is delayed due to deficient formation of collagen.

• Poor dentine formation in children, leading to poor teeth formation

. • Gums are swollen and become spongy and bleeds on slightest pressure.

• In severe scurvy, may lead to secondary infection and loosening and falling of teeth.

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Topic 4: WATER-SOLUBLE VITAMINS Cont..1
B-COMPLEX VITAMINS

THIAMINE (VITAMIN B1) Synonyms: Antiberiberi factor, antineuritic vitamin, aneurin.

Free thiamine is a basic substance containing (a) a pyrimidine, and (b) a thiazole ring. also contains sulphur (sulphur containing vitamin).

Sources

• Plant source: Widely distributed in plant kingdom. In cereal grains, it is concentrated in outer germ/bran layers (e.g. rice polishings) (Richest source). Other good sources are peas, beans, whole cereal grains, bran, nuts, prunes, etc. Whole white bread is a good source.

• Animal source: Thiamine is present in most animal tissues. Liver, meat and eggs supply considerable amounts. Ham/pork meats are particularly rich. Milk has low concentration, but a good source as large quantities are consumed.

METABOLIC ROLE

Biological active form is Thiamine pyrophosphate (TPP). Acts as a coenzyme in several metabolic reactions.

• Acts as coenzyme to the enzyme pyruvate dehydrogenase complex (PDH) which converts pyruvic acid to acetyl-CoA (oxidative decarboxylation)

• Acts as a coenzyme to α-oxoglutarate dehydrogenase complex and converts α-oxoglutarate to succinyl-CoA (oxidative decarboxylation). α-oxoglutarate dehydrogenase α-Oxoglutarate Succinyl-CoA TPP.

• Also acts as a coenzyme with the enzyme Transketolase in transketolation reaction in HMP pathway of glucose metabolism. Transketolase Ribose-5-P + xylulose-5-P TPP

Sedoheptulose-7-P + Glyceraldehyde-3-P

•B 1 is also required in amino acid Tryptophan metabolism for the activity of the enzyme Tryptophan pyrrolase.

• Also acts as a coenzyme for mitochondrial branched-chain α -ketoacid decarboxylase which catalyses oxidative decarboxylations of branched-chain α-ketoacids formed in the catabolism of valine, Leucine and Iso-leucine

DEFICIENCY MANIFESTATIONS:

Beriberi

The deficiency of thiamine produces a condition called beriberi. It is characterised by

• CV manifestations: These include palpitation, dyspnoea, cardiac hypertrophy and dilatation, which may progress to congestive cardiac failure.

• Neurological manifestations: These are predominantly those of ascending, symmetrical, peripheral polyneuritis.

. • GI symptoms: Amongst these, anorexia is an early symptom. There may be gastric atony, with diminished gastric motility and nausea; fever and vomiting occur in advanced stages.

RIBOFLAVIN (VITAMIN B2) Synonyms: Lactoflavin, ovoflavin, hepatoflavin.

• It is an orange-yellow compound containing, – A ribose alcohol: D-Ribitol – A heterocyclic parent ring structure Isoalloxazine (Flavin nucleus). 1-Carbon of ribityl group is attached at the 9 position of isoalloxazine nucleus. Ribityl is an alcohol derived from pentose sugar Dribose.

Food Sources

• Plant sources: High concentration occurs in yeasts. Appreciable amount present in whole grains, dry beans and peas, nuts, green vegetable. Germinating seeds, e.g. grams/Dals are very good source.

• Animal source: Liver, kidney, milk, eggs, and Crab meat, which has high content.

METABOLIC ROLE

FMN and FAD act as coenzymes in various H-transfer reactions in metabolism. The hydrogen is transported by reversible reduction of the coenzyme by two hydrogen atoms added to the ‘N’ at positions 1 and 10, thus forming dihydro or leucoriboflavin.

There is no definite disease entity. Deficiency is usually associated with deficiencies in other B-vitamins

NIACIN (VITAMIN B3) Synonyms: Nicotinic acid, P-P factor, Pellagra-preventing factor of Goldberger.

It is chemically Pyridine3-carboxylic acid. Amino acid tryptophan is a precursor of nicotinic acid in many plants, and animal species including human beings

In tissues: Occurs principally as the amide (nicotinamide, niacinamide). In this form it enters into physiological active combination

Food Sources

• Animal source: Liver, kidney, meat, fish

• Vegetable source: Legumes (peas, beans, lentils), nuts, certain green vegetables, coffee and tea. Nicotinamide is present in highest concentration

In tissues, nicotinamide is present largely as a “dinucleotide”, the pyridine ‘N’ being linked to a D-ribose residue.

Two such neucleotide active forms are known:

• Nicotinamide adenine dinucleotide (NAD+) other names are: DPN+, coenzyme-I, cozymase, or codehydrogenase.

Metabolic Role

• The coenzymes NAD+ and NADP+ operate as hydrogen and electron transfer agents by virtue of reversible oxidation and reduction.

• Function of NADP+ is similar to that of NAD+ in hydrogen and electron transport. The two coenzymes are interconvertible.

Deficiency Manifestations

Pellagra:

Nicotinic acid deficiency produces a disease called pellagra (Pelle = skin; agra = rough)

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Topic 4: WATER-SOLUBLE VITAMINS Cont..2
PYRIDOXINE (VITAMIN B6)

• Pyridoxol (Pyridoxine): also called as Adermin is chemically 2-methyl-3-OH-4, 5-di (hydroxymethyl) pyridine.

• It occurs in association, perhaps in equilibrium, with an aldehyde-Pyridoxal and an amine Pyridoxa mine form. All three forms exhibit vitamin B6 activity.

Biological ‘active’ forms of the vitamin are: • Pyridoxal-PO4 , and • Pyridoxamine-PO4

Food sources:

Rich sources of the vitamin are yeast, rice polishings, germinal portion of various seeds and cereal grains and egg-yolk. Moderate amounts are present in liver, kidney, muscle, fish. Milk is a poor source. Highest concentration occurs in royal jelly

Metabolic Role

Pyridoxal P acts as a coenzyme, involved with metabolism of amino acids.

• Cotransaminase: It acts as a coenzyme for the enzyme transaminases (aminotransferases) in transamination reaction.

• Codecarboxylase: It acts as coenzyme for the enzyme decarboxylases in decarboxylation reaction.

• Acts as coenzyme for deaminases (dehydrases): Catalyses non-oxidative deamination of OH-amino acids viz., serine, threonine, etc.

• Coenzyme for kynureninase: In tryptophan metabolism, pyridoxal-P acts as a coenzyme for the enzyme kynureninase which converts 3-OH-kynurenine to 3-OHanthranilic acid which ultimately forms nicotinic acid.

• Transulfuration: It takes part in transulfuration reactions involving transfer of –SH group, e.g. Homocysteine + Serine → homoserine + cysteine.

• As coenzyme for desulfhydrases: It catalyses non-oxidative deamination of cysteine in which H2S is liberated

Deficiency Manifestations

Clinical manifestations attributed to vitamin B6 deficiency include,

• Epileptiform convulsions in infants: have been attributed to pyridoxine deficiency which is related to lowered activity of Glutamic acid decarboxylase.

• Pyridoxine responsive anaemia.

• Pyridoxine responsive anaemia

FOLIC ACID GROUPS (VITAMIN B9)

• The designation “folic acid” is applied to a number of compounds which contain the following groups: –A pteridine nucleus (pyrimidine and pyrazine rings)

Active “coenzyme” form of the vitamin is the reduced tetrahydroderivative, Tetrahydrofolate F.H4, obtained by addition of four hydrogens to the pteridine moiety at 5, 6, 7 and 8 position.

Food Sources

It is found abundant in liver, yeast, kidney and green leafy vegetables. Spinach and cauliflower are also good sources. Other good sources are: meat, fish, and wheat. Fair sources: milk, fruits.

The folic acid coenzymes are specifically concerned with metabolic reactions involving the transfer and utilisation of the one carbon moiety (C1

Deficiency manifestations:

Bone marrow shows: Arrested development of all elements: erythroid, myeloid and thr

·         A macrocytic type of anaemia at times with normoblasts, erythroblasts, and megaloblasts.

·         Granulocytopenia , occasionally with myelocy                              ,Thrombocytopeniaombocytes.

VITAMIN B12 (CYANOCOBALAMINE)

It is an antipernicious anemia factor, extrinsic factor of Castle, animal protein factor.

Biological active forms are cobamide coenzymes, act as coenzyme with various enzymes.

Vitamin B12 is required as a coenzyme for the conversion of L-methyl malonyl CoA to succinyl-CoA. The reaction is catalyzed by the enzyme

Deficiency Manifestations
It causes adults, juvenile, and pernicious congenital anemia

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Topic 1: PEPTIDES AND PROTEINS
Interesting Peptide in Biological Systems:

1.         Glutathione

a.   Tripeptide of glutamate, cysteine, glycine
b.   Regulates oxidation/reduction reactions in cells
c.   Destroys destructive free radicals by scavenging oxidizing agents

2.         Oxytocin and Vasopressin

a.   Pituitary gland peptide hormones
b.   Nonapeptides cyclized by disulfide bond
c.   Oxytocin stimulates uterine contractions during childbirth – induces labor
d.   Vasopressin stimulates water resorption by kidneys and increases blood pressure (anti-diuretic hormone)

3.         Enkephalins and Endorphins

a.   Brain and nervous system peptides
b.   Important in control of pain and emotional states

4.         Insulin – Peptide hormone that regulates carbohydrate metabolism. Acts as signal for “fed” state. Functions to cap glucose levels in blood. Stimulates storage of glucose as glycogen among other effects.

5.         Synthetic Peptide: Nutrasweet or Aspartame

a.   L-aspartyl-L-phenylalanine methyl ester
b.   Dipeptide – 200X sweeter than sucrose and only 1 calorie/teaspoon vs. sucrose that has 16 calories/teaspoon
c.   Used in diet and low calorie foods and drinks
d.   Highly profitable
e.   If amino acids are in the “D” configuration, the peptide is bitter not sweet

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Topic 1: PROTEIN STRUCTURE
Each protein has a characteristic shape, size and function:

Classification of Proteins on the Basis of Biological Role:

1.         Structural Proteins

a.   Provide mechanical support to cells and organisms

b.   Give strength to bones, skin and tendons: collagen, elastin

2.         Enzymes

a.   Proteins that serve as biological catalysts for chemical reactions in cells

3.         Transport and Storage

a.   Carriers for small biomolecules to cellular destinations for use in metabolism or in construction of cell components

b.   Examples: oxygen, ferritin (iron in liver), lipoproteins that transport cholesterol

4.         Muscle Contraction and Mobility

a.   Actin and myosin are components of skeletal muscle

5.         Immune Proteins and other Protective Proteins

a.   Proteins used for defensive purposes

i. Example: Antibodies are proteins that bind and destroy foreign substances like viruses and bacteria

6.         Regulatory and Receptor Proteins

a.   Proteins that regulate cellular and physiological activity i.   Hormones

ii. DNA Binding Proteins – assist in regulation of protein synthesis b.   Receptors

i.   Proteins that mediate hormone signals and transmit the signal to the inside of the cell

1.   e.g. G-proteins and brain receptors

2.   Aspartame with taste receptor

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Topic 1: FOUR LEVELS OF PROTEIN STRUCTURE

-    Primary (1°)

o Linear sequence of amino acids in a protein

-          Secondary (2°)

o Local 3-dimensional structure of the PEPTIDE BACKBONE

      Ignores the conformation of the side chains

-    Tertiary (3°)

o Global arrangement of secondary structure, side chains (R groups), and other prosthetic groups (e.g. metals)

-    Quaternary (4°)

o Arrangement of multiple proteins into complexes

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Topic 2: CARBOHYDRATES

•    Carbohydrates are based upon the general formula Cn(H20)n
–    As if they were hydrates of carbon (most abundant organic molecules in our biosphere)

•    Functions of Carbohydrates
–    Energy source (glucose)
–    Energy storage (glycogen, starch)
–    Carbon source (pyruvate used to make Ile, Leu, Val, Ala)
–    Structure/Protection (chitin, cellulose, connective tissue)
–    Recognition/Signaling (Antibodies used for immune system recognition)
–    Can be attached to other macromolecules (glycoproteins and glycolipids)

•    Classes of carbohydrates
–    monosaccharides (simple sugars like glucose)
–    disaccharides (sucrose)
–    trisaccharides
–          polysaccharides (oligosaccharides) (starch, cellulose, glycogen) – long chains of monosaccharides; chains of monosaccharides bridged through oxygen atoms – can be thousands long and can be branched

•    Formula Cn(H20)n only holds for monosaccharides
–one H2O is eliminated when sugars are linked together to form disaccharides or higher polymers

•    Common monosaccharides contain from 3 to 6 carbon atoms
–    these sugars are called trioses, tetroses, pentoses, hexoses

•    Monosaccharides are either aldehydes or keto
–   aldoses or ketoses

•           Combining these terms describes the essential structure of sugars

–    glyceraldehyde is an aldotriose
–    glucose is an aldohexose
–    fructose is a ketohexose

•    For Aldoses and Ketoses – the name is based on the location of the carbonyl (C = O)

•    The simplest KETOSE is DIHYDROXYACETONE
-    Contains a KETONE
-    Does NOT contain a chiral center
-          Only monosaccharide that DOES NOT have a chiral center

•    Every other monosaccharide has at least one.

•    A sugar with n chiral centers can exist in 2n different forms

•    The simplest ALDOSE is GLYCERALDEHYDE
Contains an ALDEHYDE (yellow) Contains a CHIRAL center:
-    Carbon with 4 different groups bonded to it
-Because of the chiral center, has two ENANTIOMERS – non-superimposable mirror images

•    Called the D and L forms

•    Almost ALL sugars are D
•     Isomers = same chemical formula, different structure
•     Epimers = isomers that differ at only one Carbon
•     Enantiomers = isomers that are mirror images (D and L)
•     Anomers = isomers that differ only at keto-/aldo carbon

•These are the Fischer Projections for the two isomers of glyceraldehydes: Tell us stereochemistry. Tetrahedral carbon represented by two crossed lines.

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Topic 2: Carbohydrates Cont..1
Rules:
-    Carbons are numbered from the top
-          Most oxidized C (one with the most number of bonds to O goes at top (C1)
-    Last carbon will ALWAYS be part of a CH2OH group (Not CHIRAL)
-    Vertical lines go into the page
-    Horizontal lines come out of the page
-    Crosses can also be the carbons
-Stereochemistry of the last CHIRAL carbon (2nd to last C in chain) determines the stereochemistry of the sugar
•    If –OH is to the RIGHT  D-isomer
•    If –OH is to the LEFT  L-isomer
•There are 15 D-aldose sugars of 3-6 carbons in length that have multiple chiral centers
•Note ALL have –OH to the right in the 2nd to last position (last chiral centers)  D-aldoses
•Note how the positions at other chiral centers change
•All members of a row are DIASTEREOMERS of each other
•D-glucose, D-mannose and D-galactose are the most abundant aldohexose monosaccharides
•   D-mannose and D-galactose differ stereochemically from D-glucose at only 1 chiral center
o Therefore D-mannose and D-galactose are EPIMERS of glucose
o D-galactose is a C-4 Epimer of D- glucose

Same rules apply for KETOSES:

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Topic 2: Carbohydrates Cont..2
KNOW D-FRUCTOSE:

-    A common sugar

-    A ketohexose

-    Sweetest of all sugars

-    Similar in structure to D-glucose

Enantionmers of Fructose: Note, in enantionmers the positions of ALL –OH
change, not just the 2nd to last!

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Topic 2: Carbohydrates Cont..3
CYCLIZATION OF MONOSACCHARIDES

-    In solution, monosaccharides are cyclic – especially C5 and C6 sugars

-Drawn as HAWORTH PROJECTIONS – Show all hydroxyls, oxygens, and hydrogens – no carbons

-   TWO CASES OF CYCLIZATION:

-   HEMIACETALS:

Carbonyl reacting with hydroxyl group  addition product called hemiacetal. Carbon center bonded to one R-group, a H atom, an –OH and an –OR’.
o In this example, C1 carbonyl group (aldehyde) interacts with alcohol on C5 to form a six membered ring, with C6 above the ring structure.

o Reaction called an ALDOL CONDENSATION

o Form a 5 or 6 membered ring

o The C1 carbonyl carbon becomes a new chiral center – a new C1 hydroxyl

o New C1 hydroxyl = anomeric carbon
•    The carbonyl carbon in the straight chain form

•    Carbon bonded to both the ring oxygen and a hydroxyl group in the cyclic form

o Hydroxyl group is either above or below the ring – two forms α (alpha) and β (beta)

•    For D-sugars:

•      α -anomer: hydroxyl group BELOW ring (down)

•           β -anomer: hydroxyl group ABOVE ring (up) “Beta get up for breakfast”
ONLY ANOMERIC CARBON –OH is designated α or β
-    Going from FISCHER PROJECTIONS to HAWORTH:

-    Numbering remains the same for the carbons
-    If –OH is on the right  points DOWN in Haworth
-    If –OH is on the left  points UP in Haworth

-    Terminal CH2OH ALWAYS points UP relative to anomeric carbon in D sugars
a = axial position  bonds that extend straight above or below the plane of the ring e = equatorial position  bonds that are pointed outward from the ring
Chair is the most STABLE conformation. Hydrogens are axial and larger substituents are equatorial – less opportunity for steric interactions.

2.       HEMIKETALS

Hemiketal functional group includes a carbon center with 2 R-groups, an –OH and and an –OR’’ group.

•    Formed when C5 hydroxyl interacts with C2 carbonyl of a ketose

•    Example: D-fructose cyclization
- Lone pair of electrons on –OH at position C5 attacks carbonyl at C2 forming the ring
- Anomeric carbon is C2
- Hemiketals also have α and β anomers
-    Depends on stereochemistry of –OH at C2
o Down = alpha
o Up = beta

- Note: Also have a CH2OH at C1 – becomes the other group off of the anomeric C2 carbon

IMPORTANT
-      Both condensations of hemiacetals and hemiketals are freely reversible
-           In solution, there is rapid, free interconversion between the α and β anomers that necessarily passes through the open chain form
-      Process called MUTAROTATION

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Topic 2: Carbohydrates Cont..4
MUTAROTATION

Mutarotation = interconversion between the α and β anomers

[Almost all monosaccharides in solution are in cyclized form] anomeric carbon

–    REACTIONS OF MONOSACCHARIDES
Because the cyclic and linear forms of aldoses and ketoses interconvert readily, these sugars undergo reactions typical of aldehydes and ketones.
o Biologically relevant reactions
o Those used to identify carbohydrates
o 1.         OXIDATION-REDUCTION
• Complete OXIDATION of sugars supplies energy
• Sugars that can be oxidized are called reducing sugars
• Contain a free aldehyde group (found in open chain form)
• A 2-ketose will open to give its 2-ketone form, which can tautomerize to an aldehyde and then be oxidized
• All monosaccharides are reducing sugars
o Aldehyde in aldoses readily oxidized
o Ketoses isomerizes and get oxidized
•           OPEN CHAIN FORM OF SUGAR GETS OXIDIZED
•           Due to equilibrium between open and cyclic form, more open chain form will be produced once the other has reacted.

o Therefore, sugars MUST be able to open and close in ring form
o Therefore, reducing sugars MUST have a free ANOMERIC CARBON
o Free anomeric carbon = REDUCING END
o Sugars tested as reducing sugars in a test called the TOLLENS TEST
o Silver ammonia complex ion is the oxidizing agent (gets reduced)
o Sugar to be tested is the reducing agent (gets oxidized)

o DEMONSTRATION: The Silver Mirror Test
o REMEMBER: The “Reducing End” of a sugar is the end with a free anomeric hydroxyl group

The most reactive –O
Another oxidation carried
out by an enzyme: Class of enzymes = Dehydrogenases

Sugar gets oxidized and the cofactor gets reduced. Oxidation of glucose:

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Topic 2: Carbohydrates Cont..5
DISACCHARIDES

-    Formed between monosaccharides via a glycosidic bond

-    involves OH of anomeric carbon and any other OH

-    In the reaction, lose elements of H2O

-    α (1→4)

-    α = alpha = configuration of anomeric carbon

-    1 = number of anomeric carbon

-    →4 = denotes other carbon involved in glycosidic bond

-    Anomeric carbon gets fixed/locked into either α or β configuration (can’t mutarotate)

-           Having lots of variation in monosaccharides and variation in how they are linked leads to many different disaccharides

-    For example:
-    General format to describe a disaccharide is:
1st Sugar name – OH type (Carbon # 1st sugar – Carbon # 2nd sugar) – 2nd Sugar name
(α or β )
[only if at anomeric carbon]

-    Should know the structures of:
-          Cellobiose
-          Sucrose
-          Lactose

-          Maltose
-   Cellobiose
o 1st glucose is β-linked to the 4th carbon of another glucose
o Not branched, on average has 10,000 – 15,000 glucose molecules
o Very long straight chains that can bundle of parallel chains = fibrils
o glucose β(14)glucose
o β points up so draw the bond as curved or wavy line – Up from C1 and down from C4
o forms planar, crystalline structure through lots of inter- and intra-molecular hydrogen bonding
Linked with oxygen
Structural Polysaccharide

Cellulose
o Cellobiose is the repeating unit in long polymers of cellulose, the major structural component of plants especially wood and plant fibers

o Humans do NOT have the capacity to digest cellobiose or cellulose

•           Can’t digest cellulose because we lack the enzyme cellulase that breaks β(14) linkages between glucose monomers

o Ruminant animals (cattle, deer, giraffes, camels) CAN digest! Bacteria live in the rumen in GI tract and secrete cellulase.

o Termites also have bacteria in digestive tract that secrete cellulase to digest wood fibers.

Another Structural Polysaccharide: CHITIN

•    Cellulose-like homopolymer

•   Exoskeleton of insects and crustaceans, cell wall of many fungi

•           β(14) linked glucose derivative: N-acetylglucosamine (glucosamine with an acetyl group linked to the amino group at C2)

•           Modification allows for increased hydrogen bonding between adjacent polymers, giving the chitin-polymer matrix increased strength

Lactose:

-    Galactose in a β(14) linkage with glucose

-           Galactose is converted by the body to glucose and glucose used for energy

-    Lactose IS digestible by most humans
-    Found in dairy products
-           Enzyme LACTASE present in small intestine hydrolyzes lactose to galactose and glucose.
-5% of people from Scandinavia and 90% of Asian adults suffer from lactose intolerance – deficiency in enzyme lactase.
-    For those with the deficiency:
o Lactose accumulates in small intestine
o Degraded by intestinal bacteria producing
CO2, hydrogen gas, and organic acids
o Presence of excess undigested lactose is harmful as well
o Both cause symptoms: Bloating
Nausea Cramping Diarrhea
o Treatment:
•           Avoid products containing lactose (dairy products)
•           Use commercial products to hydrolyze lactose before consumption
•    Add enzyme called β-galactosidase (e.g. lactaid milk)

-   Sucrose:
o Glucose in α,β (12) linkage with fructose (50% glucose:50% fructose)
o NEEDS BOTH DESIGNATIONS! Both ANOMERIC CARBONS are involved in the linkage.

•    Anomeric carbon (C1) on glucose is linked to the anomeric carbon (C2) on fructose

•    BOTH anomeric carbons are involved in glycosidic bonds

•    Therefore, sucrose is NOT a reducing sugar

•          Sucrose, glucose and fructose most common natural sweeteners
-    Corn syrup: glucose-heavy syrup made from corn starch - contains no fructose
-           High fructose corn syrup: Glucose in corn syrup is converted to fructose enzymatically. Mixed with corn syrup (glucose) to produce HFCS containing 55% fructose and 42% glucose. Larger sugar molecules called higher saccharides make up the remaining 3 percent of the sweetener.
-           In HFCS, the fructose and glucose are free and can be used by your body immediately. In sucrose they are linked by the glycosidic bond and must first be broken down enzymatically by your body before it can be used.
•           Humans can synthesize all the different sugars we need from glucose – we DON’T have to make a particular kind
-    Also, our bodies can make glucose in many, many different ways
-    So, there is no explicit dietary need for sugars as long as we get enough calories
-    Sugars are so important that the body won’t leave anything to chance!
-    Remember: for amino acids, there are essential and non-essential amino acids

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Topic 2: Carbohydrates Cont..6
POLYSACCHARIDES
o Two main functions:
•    Energy Storage
•    Structure

o    STORAGE POLYSACCHARIDES:
-    STARCH
o Found in chloroplasts of plant cells – especially abundant in potatoes, corn and wheat

o Mixture of 2 types of GLUCOSE POLYMERS
• Amylose
•    Linear, unbranched chain of α(14) D-glucose molecules
•    Disaccharide repeating unit = MALTOSE
•    Each amylase has 2 ends:
•           Non-reducing End (Glucose molecule with free –OH on C4)
• Reducing End (Glucose molecule with free –OH on
C1 – anomeric carbon
• Forms a coiled, relatively compact helical structure
(~6 glucoses/turn)
AMYLOSE

D-GLUCOSE REPEATING UNITS = α (1 4) glycosidic linkages
Structure of the main backbone of amylose, amylopectin and glycogen
One turn of the helix has SIX glucose units

Amylopectin
•        Main backbone is amylose (linear) with D-glucose molecules in α(14) linkage
•        Also has BRANCHES: Connect to backbone and to each other by α(16) linkages
•        Branch points every 25-30 glucoses
•        Has ONE reducing end
•        Has many non-reducing ends

DEGRADATION OF STARCH/AMYLOPECTIN

-Amylose [α(14) linked glucose] is degraded by enzymes called AMYLASES in the mouth and intestine to yield maltose and glucose
-    Acid in your stomach also helps break down linkages
-    Maltose (diglucose) is further degraded to 2 glucoses by maltase in the intestine
-    All glucose is then absorbed by the body and used to make cellular energy
-Additional enzymes are needed to hydrolyze the α(16) linkages between glucoses at the branches – called a “debranching” enzyme

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Topic 2: Carbohydrates Cont..7
GLYCOGEN: Animal carbohydrate storage
FUNCTION:
•   All cell types: Glucose reserve, ATP from glycolysis
•   Skeletal Muscle

o Used to generate ATP during anaerobic muscle contraction
        Glycogenolysis (degrading glycogen) and glycolysis (degrading glucose)
active together
•   Liver: The 1 source of glucose for maintaining blood glucose
        Glycogen degradation tied to glucose synthesis
•   Glycogenolysis and gluconeogenesis
•   Stored in liver and muscle as granules or particles
        Up to 10% of liver mass and 1-2% muscle mass
•   Branched glucose polysaccharide
o    Chains of glucose units
o    Similar in structure to amylopectin
o    Backbone linked by α -1,4 bond (like amylose)
o    Have α -1,6 branches every 8-10 residues (like amylopectin with more branches)
o    Has one reducing end and many non-reducing ends

GLYCOGEN (pink granules) IN LIVER CELLS

STARCH                                                     GLYCOGEN
Branches every 25 units                                Branches every 8-10 units

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Topic 2: Carbohydrates Cont..8
•   GLYCOPROTEINS

–    Oligosaccharides can also be attached to proteins

–    Through glycosidic linkages to serine, threonine or asparagines

•    O-glycosidic linkages to Ser or Thr

•    N-glycosidic linkages to Asn

Different configurations of sugars on proteins

Great diversity!

FUNCTIONS OF OLIGOSACCHARIDES ON PROTEINS:

•    Influence structure, folding and stability of protein

•    May determine the lifetime of a protein (mark protein for age)

•    Serve as markers to identify a cell type

•    When glycosylated proteins are at the cell surface:

o Can modulate cell-cell interactions

Changes in carbohydrate content may influence contact inhibition of cells

o Can modulate cell – molecule interactions (e.g. hormone w/receptor)

o Can serve as antigenic determinants (how antibody recognizes the protein) on proteins

•           e.g. The difference between blood types is due to glycosylation of red blood cell proteins

BLOOD TYPES AND GLYCOSYLATION

      Presence or absence of the terminal carbohydrate is genetically determined and determines the blood type.

 Blood plasma contains antibodies against foreign blood-group antigens that aggregate the foreign blood cells

        Type A blood has antibodies that recognize B sugars

        Type B blood contains antibodies against A sugars

        Type O blood has antibodies against both A and B sugars (universal donor)

        Type AB blood contains neither antibody (universal acceptor)

        Incompatible blood types cause precipitation of RBCs, block blood flow in organs and can cause death

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Topic 3: Lipids
LIPIDS

•   Very important biomolecules

•   Insoluble in water

•   Soluble in organic solvents and other lipids

FUNCTIONS OF LIPIDS

•   Storage molecules for ENERGY (fats and oils)

o Can get lots of energy from a fat

o Stored in adipose tissue

•   Structural components of cellular membranes

•   Protective molecules (waxes)

•   Hormones and vitamins

•   Intracellular messengers

•   Pigments

•   Insulation

FOUR MAIN CLASSES OF LIPIDS

1.   Triacylgylcerols (TAGs) – Storage Lipids (non-polar)

o Also known as

triglycerides

2.   Phosphoacylglycerols – Membrane Structural Lipids (polar)

3.   Sphingolipids – Membrane

Structural Lipids (polar)

These three have the basic structure of a FATTY ACID

4.   Non-saponifiable Lipids – Steroids, hormones, cholesterol

o Based on a fused ring structure rather than fatty acids

FATTY ACIDS (FA)

•   Long chain carboxylic acids

o 12-20 hydrocarbon LINEAR chains (most even #)

o No hydrogen bonds form between the carboxylic acid functional group

        Fatty Acids interact through HYDROPHOBIC INTERACTIONS

o By nature, fatty acids are AMPHIPATHIC – have both hydrophilic and hydrophobic parts

o Often have double bonds

o TWO TYPES

        Saturated – hydrocarbon has NO double bonds

        Unsaturated – Hydrocarbon chain has ONE or MORE DOUBLE BONDS

•   Double bonds are “cis” configuration

•   Cause a kink or bend in the chain

Saturated                          Unsaturated

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Topic 3: FATTY ACIDS
o Lipids can contain many different fatty acids

o Fatty acid chain length and degree of unsaturation affect

        Melting point of lipids

        Fluidity of lipids

o SATURATED FATTY ACIDS

        Pack close together

        Less fluid (FAs can’t move as freely)

        Higher melting temperature because it takes more energy to break interactions

        Likely to be solids at room temperature

o UNSATURATED FATTY ACIDS

        Do NOT pack as closely

        More fluid than saturated

        Lower melting temperature than saturated

        Likely to be liquid at room temperature

NOMENCLATURE OF FA’s

-         Referred to as a system of numbers

-          # of carbons: # double bondsΔ x, y, z (position of double bonds)

-          For example: oleic acid

# of carbons is counted from the carbonyl end and includes the carboxyl carbon Double bond starts at number written, therefore between 9 and 10 in example pKa of carboxylic acid is ~4-5; therefore deprotonated at physiological pH

- Should be familiar with Table 8.1 (Edition 3) or Table Below

o Draw any fatty acid correctly if given the abbreviation and give numerical name if given the structure

o Know common names and structures of:

        Palmitic acid (26% of human fat)

        Oleic Acid (45% of human fat)

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Topic 3: TRIACYLGLYCEROLS

•    Triacylglycerols are made up from 3 fatty acids ester linked to glycerol

-           Each –OH on glycerol can react with a fatty acid

-    Start with C1  C2  C3

-    Release H2O upon formation of ester linkage

Can see from the structures that triacylglycerols (TAGs) are neutral (no ionic groups), non-polar and hydrophobic

Triacylglycerols as STORAGE LIPIDS



-    Comprise fats and oils

o OILS usually from plants

 Contain more unsaturated fatty acids – liquid at room temperature

 Except coconut oil

o FATS usually from animals

        Contain more saturated fatty acids

        Recommended: Consume more unsaturated than saturated fats

        Saturated fat leads to atherosclerosis, heart disease and cancer

        Stored in adipocytes – only function is to store fat

•   Found in oily droplets in the cytoplasm

        Rich source of energy

•           More highly reduced and not hydrated – more to oxidize and give energy!

FAT SUBSTITUTES:

-           Olestra – chemically synthesized fat (TAG) substitute

-           Mixture of sugars and fatty acids

- Not absorbed and metabolized

– therefore not caloric

-BUT, depletes the body of fat soluble vitamins and may lead to gastrointestinal distress

TRANS-FATTY ACIDS & MARGARINE

•Trans-fatty acids are found in margarines – wanted a solid butter substitute that is high in desired polyunsaturated fats

•    Take liquid corn oil and partially hydrogenate

–    Hydrogenation leads to the reduction (saturation) of cis-double bonds

•Changes some double bonds to single bonds to single bonds and transforms oil to firm but spreadable solid

–    However, hydrogenation can also produce trans-fatty acids as by-products

–Trans-fatty acids thought to raise blood cholesterol: raise LDL (bad cholesterol) and lower HDL (good cholesterol)

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Topic 3: TRIACYLGLYCEROLS Cont..1
1.       TRIACYLGLYCEROLS:

-    How are they broken down?

o Hydrolyzed into 3 fatty acids and 1 glycerol

o Physiologically in body:

Enzyme called a LIPASE present in adipocytes and intestines

o Saponification

Treat with base (NaOH) and heat to produce soaps (salts of FAs) and glycerol Used to (and still do!) boil animal fat with lye (NaOH) to make soap!

2. PHOSPHOACYLGLYCEROLS (Phospholipids; Phosphoglycerides)

-Very similar in structure to triacylglycerols except one of the alcohols of glycerol is esterified by phosphoric acid instead of a fatty acid = phosphatidic acid (PA)

-    Phospholipids are MUCH MORE amphiphilic than triacylglycerols due to

CHARGED groups at neutral pH

o Has both hydrophilic and hydrophobic regions

-    Therefore we can say that phospholipids have:

o One POLAR HEAD

o TWO NON-POLAR TAILS

-    Phospholipids can be degraded to their component parts by a family of enzymes called

PHOSPHOLIPASES

o EXAMPLE: SNAKE VENOM

Venoms of poisonous snak contain (among other things) phospholipases which cause the

breakdown of the phospholipids Western Diamondback Rattlesnake and Indian Cobra contain Phospholipase A2

Phospholipase A2 catalyzes the hydrolysis of fatty acids at the C2 Position

-    Remaining compound called lysolethicin

o “one-legged” phospholipids

o Acts as a detergent

o Dissolved membranes in red blood cells causing them to rup

Acts like a detergent that

LYSOLECITHIN:

disrupts and dissolves membranes in red blood cells

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Topic 3: Other Lipids
3. SPHINGOLIPIDS

-    Membrane lipids based on the core structure of SPHINGOSINE, a long chain amino alcohol

o Glycerol is replaced by sphingosine

Sphingolipids:

-    Much more amphiphilic than triacylglycerols

-    Sphingomyelin

o Insulates nerve axons

o Major lipid of myelin sheaths

-    Cerebrosides and Gangliosides

(glycolipids)

o Abundant in brain and nervous system membranes

o Improper degradation results in many

metabolic diseases

        Tay-Sachs Disease

•   Gangliosides accumulate

in nerve cells, brain, and spleen  Death!

        Gaucher Disease

•   Accumulation of glucocerebrosides

o Enlarged liver and spleen

o Bone pain

o Anemia

•   Deficiency in the enzyme glucocerebrosidase

4.         NON-SAPONIFIABLE LIPIDS/STEROIDS

-          Based on a fused ring system – RIGID structure

-          No ester linkages

-          Includes HORMONES (testosterone, progesterone, estrogen)

Cholesterol

o Common membrane lipid

o Almost exclusive to animal cells

o Very hydrophobic but amphiphilic

Hydrophilic group is the –OH on ring A

o Serves as the starting point for synthesis of steroid hormones

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Topic 1: Nucleotide Metabolism
Disorders of Nucleotide Metabolism: Hyperuricemia and Gout

- Gout (also called urate crystal deposition disease) is a condition characterized by hyperuricemia - high levels of uric acid

- the deposition of monosodium urate and uric acid crystals in tissues as a result of supersaturation of extracellular fluids with urate

- gout is manifested by recurrent attacks of acute inflammatory arthritis, the development of uric acid stones and renal disease

- like PKU, gout has both genetic and environment contributions

- disease has been recognized for centuries (Henry VIII, Benjamin Franklin)

Clinical features of gout

- symptomatic manifestations of gout only arise in a minority of persons with hyperuricemia, and usually only after 20-30 years of sustained hyperuricemia

1)    Acute gouty arthritis: episodes of painful inflammatory arthritis, last hours to weeks and are usually monoarticular (affects only one joint); big toe is common site

“ The patient goes to bed and sleeps quietly till about two in the morning when he is awakened by a pain which usually seizes the great toe, but sometimes the heel, ankle or instep. The pain resembles that of a dislocated bone…and is immediately preceded by a chillness and slight fever in proportion to the pain which is mild at first but grows gradually more violent every hour; sometimes resembling a laceration of ligaments, sometimes the gnawing of a dog, and sometimes the weight and constriction of the parts affected, which becomes so exquisitely painful as not to endure the weight of the clothes nor the shaking of the room from a person walking briskly therein.”

2)   Urate nephropathy: a renal disease caused by deposition of urate crystals in the interstitial space of the kidneys, can lead to kidney failure

3)   Uric acid urolithiasis: bladder stones

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Topic 1: Overview of purine metabolism
- ATP is substrate for the cellular transmethylation cycle to form S-adenosylmethionine (SAM); during cellular transmethylation, adenosine is formed and feeds into the purine degradation pathway

- salvage of purines from dietary sources

- RNA degradation

Uric Acid Homeostasis

- ionized forms of uric acid readily form salts

- in extracellular fluids in which sodium is the principal cation, 98% of the uric acid is found as the monosodium salt at pH 7.4

- crystals of monosodium urate monohydrate form in the synovial fluid when the solubility limits are exceeded

Why do we have any uric acid?

- uric acid is a breakdown product of purines (ATP, GTP, nucleic acids) and its excretion permits the necessary removal of nitrogen waste from the body

Overview of purine catabolism

Why do we have any uric acid - part 2?

- may also play a role in immunity as an adjuvant vaccination of an organism with antigen alone is likely to induce tolerance rather an immune response without the presence of an adjuvant known adjuvants: mycobacterium, LPS (act via toll-like receptors and upregulation of co-stimulatory molecules such as CD86 to induce a full T-cell response) mammalian cytosol from dying or damaged cells can be an adjuvant; in

2003 Shi and colleagues showed that uric acid is one of the cytosolic

factors (only crystalline uric acid ca

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Topic 1: Purine Metabolism
Why did evolution select for an endogenous adjuvant?
“danger signal” concept: in 1990s, Matzinger postulated that cells damaged by trauma or viral infection might need a way to signal the immune system
- programmed cell death initiates an active process of DNA fragmentation and purine degradation, leading to high urate production in the dying cell
- Hu and colleagues used a mouse model of immunologic tumor rejection to test this theory
- they observed that lowering levels of uric acid with either allopurinol or uricase led to delayed tumor rejection and treating the tumor mice with uric acid enhanced the rate of rejection

Why do humans and other primates have so much uric acid?
- unlike most mammals with serum urate levels below 2 mg/dL, primates tend to have serum urates in the range of 6 to 7 mg/dL, due to the lack of the uricase enzyme
- uricase breaks down uric acid to a more soluble component, allantoin prior to excretion
- uric acid is a weak acid that may exert antioxidant effects
- the fact that loss of uricase occurred in the same era suggests that it may have conferred a survival advantage during that period
- our ancestors in the Miocene era were mainly limited to a diet of fruits and grasses (low in sodium); this low sodium diet may have led to a hypotensive “crisis”
- loss of uricase and accumulation of uric acid might have compensated for Hypotension
- biped more dependent on blood pressure to maintain cerebral perfusion the experiment…
- rats fed a low-sodium diet then treated with oxonic acid, a uricase inhibitor
- this effect can be blocked by allopurinol, a xanthine oxidase inhibitor that reduces uric acid biosynthesis

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Topic 1: Purine Metabolism cont..
Why do some human beings have too much uric acid?
- normal range in humans: 4-7 mg/dL; higher levels known to trigger attacks
- trauma, surgery, excessive ingestion of alcohol or purine-rich foods, starvation and administration of certain drugs (diuretics, cyclosporine, etc.)
- all the causes of gout are not extrinsic; this condition is associated with many genetic disorders
* handling of uric acid by the kidneys (URAT1 and UAT1)
* overproduction of uric acid can lead to hyperuricemic
- hereditary hyperactivity of the purine synthesis enzyme PRPP (5-P-D-ribosyl-1
-pyrophosphate)

 partial deficiency of HGPR (hypoxanthine-guanine phosphoribosyl) transferase; the rate-limiting step in the purine salvage pathway
PRS superactivity and HPRT deficiency: two inborn errors associated with hyperuricemia and gout
- PRS superactivity; increased PP-Rib-P levels due to overproduction
(no apparent decreases in purine nucleotide concentrations)
- HPRT deficiency: PP-Rib-P accumulates due to underutilization of this salvage reaction
Increased PP-Rib-P availability in both cases results in activation of AmidoPRT and acceleration of purine nucleotide and uric acid synthesis

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Topic 1: Mechanisms of Hyperuricemia: increased ATP degradation
- Exercise

- Ethanol digestion

- Glucose-6-phosphatase deficiency (glycogen storage disease type I)

- Fructose-1,6-phosphatase deficiency

- Fructose infusion and hereditary fructose intolerance

Last three examples are noteworthy since they suggest that defects in carbohydrate metabolism may manifest themselves as hyperuricemia and gout

Mechanism of fructose-induced purine nucleotide degradation

Treatments for gout

- acute gouty arthritis is most commonly treated with NSAIDs, steroids or colchicine

- lowering uric acid to prevent gouty attacks can be accomplished with allopurinol but there are contraindications for this therapy including diabetes, renal insufficiency and gastrointestinal pathology

- Febuxostat: nonpurine xanthine oxidase inhibitor that mimics the action of allopurinol

- Uricase infusion: lowers serum uric acid levels and insoluble urate crystal deposits (has same problems faced by PAL - highly immunogenic, poorly tolerated, short half-life)

- pegylation of uricase shown to be effective in improving pharmacological parameters

Pathophysiology of gout: How does hyperuricemia lead to an inflammatory response to urate crystals?

-presence of crystals stimulates a two-pronged inflammatory signal

* activation of complement results in chemoattractant generation which activates and attracts bloodstream neutrophils

* vascular endothelial cells must first be activated by cytokines generated by macrophages lining the synovium (IL-1, IL-6 and TNF-α)

- new evidence indicates a role for the inflammasome in the onset of gout

- inflammasomes are structures that mediates the production of IL-1 from its propeptide form proIL-1

- proteolysis of propeptide is carried out by caspase-1 which must first be aligned on a number of scaffolding complexes

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Topic 2: Phenylketonuria (PKU)

- classic “inborn error of metabolism”

- autosomal recessive disease characterized by mutations in the liver enzyme, phenylalanine hydroxylase, encoded by the PAH gene.PAH converts phenylalanine to tyrosine

(reaction requires O2 and co-factor BH4)

- HPA or non-PKU hyperphenylalaninemia are related disorders of phenylalanine hydroxylation involving several enzymes necessary for the synthesis and recycling of co-factor for PAH,tetrahydrobiopterin (BH4)
- incidence: 1 in 10,000

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Topic 2: PKU cont'd
PKU has a multifactorial cause:

§ mutation in PAH gene (genetic)

§ exposure to dietary phenylalanine (environmental)

Clinical features of PKU

Enzyme deficiency is a primarily hepatic phenotype but major clinical presentation is abnormal brain developmentand function

- reduced higher-brain abilities (executive functions)

- neuropsychological dysfunction (imbalance of neurotransmitters)

- emotional disturbance and behavioral problems (clinical depression)

- severe mental retardation will result in untreated cases (estimated that

1% of patients in mental institutions have PKU

Subtypes of PKU, phenylalanine levels & clinical outlook

Fold increase blood [Phe]      clinical picture         treatment

    subtype              (over normal)             (brain dysfunction)      required?

classic PKU    >20      severe mental yes

(untreated)                  retardation

mild PKU                  10-15                         cognitive loss             yes

(untreated)

non-PKU                     2-8                                normal                maybe mild HPA

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Topic 2: Screening for PKU
Newborn screening for PKU

- done with a simple blood test, screening is standard in many developed countries

- resource for sampling of mutant PAH genes

- prenatal diagnosis is possible

- classification of severe and less severe forms as well as non-PKU HPA requires Phe and BH4 measurements in several body fluids

Maternal PKU

- pregnant mothers with untreated PKU can give birth to children with severe defects

- congenital malformations

- microcephaly

- severe mental retardation

- careful treatment with diet is compatible with normal outcome for fetus

Pathogenic PAH alleles

• null alleles or gene deletions (no activity)

• Vmax alleles (reduced activity)

• kinetic alleles (altered affinity for substrate or cofactor)

• unstable alleles (increased turnover and loss of PAH protein)

majority of mutations

Effects of disease-causing PAH mutations on a patient can be measured at three levels:

• proximal (enzymatic) : in vitro assay

• intermediate (metabolic) : plasma phenylalanine levels

• distal (cognitive function) : IQ tests genotype-phenotype correlations show good correlations at the proximal levels at intermediate and distal levels, phenotypes behave as complex traits suggesting the presence of “modifiers”

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Topic 2: Pathophysiology of PKU

- metabolites of PKU (i.e. phenylpyruvate) not present in high enough concentrations to be toxic

- is phenylalanine the neurotoxic agent?

1) brain protein synthesis

2) transport processes and neurotransmitter biosynthesis (tyrosine (Tyr) and tryptophan (Trp) are transported across blood-brain barrier for synthesis of the neurotransmitters, dopamine and serotonin, respectively)

Phe has higher affinity for transporter compared to Tyr and Trp

Blood-brain barrier

- hypotyrosinemia:   low [tyrosine], low neurotransmitters

(loss of biogenic amines at critical stages in postnatal brain maturation)

- decreased protein synthesis in brain (weak evidence)

- defective brain myelination (chronic and irreversible)

Potential problems with the low tyrosine theory...

• postnatal tyrosine supplementation without reduction of phenylalanine intake does not prevent mental retardation in PKU

• no consistent or pathological reduction in plasma tyrosine content in untreated PKU patients

• tyrosine supplements during treatment of PKU sufficient to increase plasma tyrosine levels do not improve neurophysiological parameters

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Topic 2: Treatment of PKU
Treatment: dietary restrictions adherence to a phenylalanine-free diet postnatally can prevent mental retardation and improve behavior in children with PKU synthetic dietary supplement needed to avoid malnutrition

Phenyl-free: Phe-free amino acid mixture, vitamins, minerals, fat (marketed by Mead Johnson)

- offensive in odor and taste

- must be continued for life

- emotional stress in PKU families

- high cost (“patient years”)

Aspartame (NutrasweetTM) is an amino acid sweetener, with two constituent amino acids, aspartic acid and phenylalanine, both commonly found in food.

Phenylalanine synthetic diet not perfect...

- produces several biological side effects due to periodic nutrient deficiencies

- needs improvement in organoleptic properties (essential fatty acids)

and nutrient composition (ratios of amino acids)

Treatment alternatives:

- gene therapy (not yet applicable)

- enzyme replacement therapy (PAL and PEG-PAL papers)

PAL: non-mammalian enzyme; degrades Phe to ammonia and trans-cinnamic acid (harmless metabolite)

Treatment of mild PKU with tetrahydrobiopterin (BH4) loading

- several recent studies suggest that BH4 can be a treatment alternative to dietary restriction of phenylalanine

Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria

N Engl J Med. 2002 Dec 26;347(26):2122-32

• out of 38 with PAH deficiency, 87% showed responsiveness to BH4

(i.e. had lower blood phenylalanine levels)

• no response in 7 patients with classic PKU

• long-term treatment with BH4 in 5 patients increased daily phenylalanine tolerance enough to discontinue Phe-restricted diet

• mutations connected to BH4 responsiveness predominantly in the catalytic domain of the protein and were not directly involved in cofactor binding

Treatment of classical PKU with BH4

- recent reports indicate that BH4 loading was also beneficial to patients with more severe forms of PKU not just mild non-PKU HPA

38 US PKU patients were given single dose of BH4 and Phe levels were monitored

58% responded at 24 h (>30% decrease in Phe levels); some who responded favorable were clinically described with classical PKU

mutant PAH responds with increase in the residual enzyme activity following

BH4 administration

- increased stability

- chaperone effect (better folding)

- correction of mutant Km

KuvanTM: synthetic form of BH4 that is approved in Europe for treatment of non-PKU HPA

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