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ENZYMOLOGY

Outline
Classification Co-factors and coenzymes Enzyme kinetics Regulation of enzyme activity Clinical applications of control of enzyme action Assignment

Lecture 1

Dr. S. Mukanganyama Biomolecular Interactions Analyses Lab. Department of Biochemistry University of Zimbabwe MBCHB & BDS1 2011

TWO FUNDAMENTALS OF LIFE

Reproduction with high fidelity Ability to catalyse reactions efficiently and selectively

ENZYMES
Specialised proteins that accelerate (catalyse) chemical reactions. Cell physiology, growth and behaviour(life) depends on enzyme action. Lower activation energy (Ea), an activation barrier for the reaction being catalysed Catalyst accelerate both forward and reverse reactions. therapeutic targets to combat diseases of either genetic or pathogenic origin

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Enzymes differ from ordinary chemical catalysts:

Higher reaction rates (about 106-1012 greater) Milder reactions conditions with temperatures below 100 oC, atmospheric pressure and nearly neutral pHs. Greater reaction specificity -substrates and products. Capacity for regulation - allosteric substances or by covalent modification. Very large molecules vs. substrates or functional groups catalysed Bind substrates through noncovalent forces such as Van der Waals, electrostatic, hydrogen bonding, and hydrophobic interactions. Catalysis at active site of the enzyme

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Enzyme Nomenclature
International Union of Biochemistry and Molecular Biology, (IUBMB). A systematic basis for naming and classifying enzymes All enzymes placed in six major classes, each with a subclass, based on the type of reaction catalysed. Each enzyme is assigned a four digit classification number and a systematic name, which identifies the reaction catalysed e.g. ATP + D-glucose ADP + D-glucose 6phosphate The formal systematic name of this enzyme is ATP: glucose phosphotransferase, which indicates that it catalyses the transfer of a phosphate group from ATP to glucose. Placed in class 2 of Table 1 Most enzymes catalyse the transfer of electrons, atoms, or functional groups. Given code numbers, and assigned names according to the type of transfer reaction, and the group donor, and the group acceptor. International (IUBMB) classification of enzymes based on the reactions.

The enzyme active site

The main features: A small portion of the total volume of the enzyme. A three-dimensional entity. Specificity of binding depends on the precisely defined arrangement of atoms in the active site giving a direct fit or an induced fit. Substrates are bound to enzymes by relatively weak forces. Clefts (indentations) or crevices on the surface of an enzyme that are complementary in shape to the substrates

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Enzyme action
Need for cofactors -function as the enzymes "chemical teeth". E.g. Zn 2+ required for catalytic activity of carboxypeptidases A, or organic molecules known as coenzymes e.g. NAD+. Prosthetic groups, cofactors permanently associated with their protein often by covalent bonds. Holoenzyme -A catalytically active enzyme-cofactor complex Apoenzyme is the protein part of the enzyme.
apoenzyme (inactive) + cofactor holoenzyme.

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Mechanisms of enzyme action

Equation for any enzyme-catalysed chemical reaction can be written in the generalised form
enzyme(cofactor)

Substrates (S)
invertase (b-fructofuranosidase)

Products (P)

e.g. sucrose + H2O

glucose + fructose

Major factors in enzyme catalysis

Orbital steering-enzyme binds the substrate (S) (and cofactor) in such a way that the susceptible bonds are (a) in close proximity to the catalytic group on the active site (the entropy effect) and (b) so oriented in relation to the catalytic group that the transition state is readily formed Formation of unstable covalent intermediate that more readily undergoes reaction to form products. General-acid or general-base catalysis-Provide functional groups capable of acting as protein donors or acceptors, the enzyme may bring about

May induce strain or distortion in the susceptible bond of the substrate, making the bond easier to break.

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CHEMICAL KINETICS
S P Rate = K+1[S]
O H
+ O H

S+S P Rate = K+1[S]2 S+A

K+1

K+1

O H

+ O H

C N H

O 3 H

3 C H

O H m

e n i h p r o

O H

O H

O H

H N

3 C O H

3 C O H

g r e W , y l n a p w o h

d u y h w m a r c o t s e l n i

S C f n v p o W . w d u r s l a i m h c e t

H P w D l m h o i t c d n a s e r

g d v p s n o a r D m e S C h t i w

i h r m o p a h g i p c s m r f u o y d t n e . w a

w u o y f I r d e k s a

m a x k d y c i l o p n r u t e

k r o w u f n i 3 0 8 1 l c s a e

S C f s v g n l i a m e r o 0 3 7 5

t w D h s e g d i r b m a c @ o f

s e u q n , y d 0 3 a m o c . t f

For a reaction in which a single-reactant molecule S is converted to P, the rate of reaction is proportional to the concentration of S. First P order reaction. K first order reaction constant.

Rate = K+1[S][A] S
K+1 K-1

Keq =K+1/K-1 = [P]/[S]

Enzyme-catalysed reactions
More complex + characterised by measuring the initial rate of the reaction, Vo, which is the reaction rate at time zero. Reaction will start at a high rate and slow down over time for several reasons: Substrate will be used up and the reaction rate will slow down as each enzyme molecule spends more time diffusing through the solution before it collides with a new substrate molecule. As the reaction proceeds, product will actually accumulate which may tend to inhibit the enzyme. The enzyme molecules may gradually loose activity owing to random hydrolysis, denaturation or endproduct inhibition.

The primary and overriding interest of enzymes, however, is their connection with life. Of all the multitudinous chemical processes in the living cell on which life depends, there is scarcely one which is not due enzyme catalysis; there can be no life without enzymes. (Dixonn and Webb, 1979)

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Effect of substrate
At Low substrate concentrations, a graph of reaction velocity against substrate concentration will be straight line. For enzyme-catalysed reaction, even with only one substrate and one product, this graph will curve at higher substrate concentration, and will level off at very high substrate concentration. i.e. enzyme has become "saturated" and the reaction rate reaches a constant value - maximum reaction rate - Vmax.

Enzyme Kinetics
Relationship between enzyme (E) and substrate (S) to give a product (P) can be described as follows:
K 1 K 2

S
K -1

ES

P + E

(i)

Where ES is the enzyme-substrate complex.

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