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Enzymes

Higher reaction rates

Milder reaction conditions

Greater reaction specificity

Zymogen

Freed Ile activates the enzyme


Enzymes
Enzymes
Enzyme substrate specificity

Enzyme are stereospecific

Enzyme vary in geometric specificity


Enzymes
Cofactors

Cosubstrate
Enzymes
Free energy of activation ΔG‡ of a one step reactions
Enzymes
Free energy of activation ΔG‡ of a two step reactions
Enzymes
Catalyst affect ΔG‡ of a reaction
Enzymes
Catalytic Mechanisms

Acid-base catalysis

Covalent catalysis

Metal ion catalysis

Proximity and orientation effects

Preferential binding of the transition state complex


Enzymes
Acid-base catalysis (Keto to Enol tautomerization)
Enzymes
Acid-base catalysis
Enzymes
Acid-base catalysis RNase A
Enzymes
Covalent catalysis

Nucleophile

Unprotonated amino group of Lys, immidazole group of His, COOH of


Asp and OH of Ser; and several coenzymes Thymine-PP and
Pyridoxal phosphate
Enzymes
Covalent catalysis
Enzymes
Metal ion catalyst
Binding to substrate and orient them properly for reaction
Oxidation-reduction reactions through reversible changes in the metal’s
oxidation state
Zn2+ polarises a water molecule
Stabilizing or shielding negative charges to form OH-

Carbonic anhydrase
Enzymes
Proximity and Orientation

Covalent bond formation improves proximity and orientation of reactants


Enzymes
Preferential binding of the transition state

Transition state analogs are enzymes inhibitors


Enzymes
Lysozyme
Enzymes
Lysozyme
Lysozyme

Glu35 acts
as an acid
catalyst

Asp52 acts as
a covalent
catalyst
Serine Proteases
Active site residues identification by chemical labeling
Serine Proteases

Chymotrypsin
Serine Proteases
Serine Proteases
Serine Proteases
Serine Proteases
Stabilization of the transition state
Tetrahedral intermediate
Oxyanion

H-bond
Strong H-bond with
shorter bond lengths
Low barrier H-bonds (LBHB)

LBHB exist in nonaqueous active sites of enzymes, but not in water


Tetrahedral intermediate resembles enzyme-inhibitor complex
Structure of tetrahedral intermediate is known
Enzyme kinetics
Biological processes can be described by elementary reactions

Consider the elementary reaction

Velocity of a first order reaction


unit of k =
For a bimolecular reaction

Velocity of a second order reaction unit of k =

For a bimolecular reaction

Velocity of a second order reaction


Enzyme kinetics
For a first order reaction

y = mx + b

Half-time /
Half-life
Enzyme kinetics
For a second order reaction

A plot of 1/[A] vs. t would be linear

t1/2 depends on initial reactant concentration

For a second order reaction


Keep one of the reactant at very high concentration so that reaction depends on the
concentration of the second reactant and becomes a pseudo-first-order reaction
Enzyme kinetics
Michaelis-Menten equation assumes that [ES] maintains a steady state

β-fructofuranosidase
At very high concentration of Sucrose the reaction
rate is zeroth order with respect to sucrose

Difficult to integrate
Enzyme kinetics
Michaelis-Menten equation assumes that [ES] maintains a steady state

Assumption of equilibrium

The first step of the reaction reaches equilibrium

ES complex is known as Michelis complex

Assumption of steady state

[ES] remains constant during much


of the reaction
George Briggs and John
Haldane

[ES] and [E] are not measurable


but [E]T is known
Enzyme kinetics
With steady state assumption

Michaelis constant
Enzyme kinetics
Initial velocity of the reaction Vo

Maximal velocity of the reaction Vmax

At high substrate concentration when the enzyme is saturated in ES form

Michaelis-Menten equation

A rectangular hyperbola similar to


binding of Myoglobin to O2

At KM = [S], Vo = Vmax/2

KM is the substrate concentration at


which the reaction velocity is half-
maximal
Enzyme kinetics
Catalytic constant

is a measure of catalytic efficiency of the enzymes


Enzyme kinetics

Lineweaver-Burk Plot

Double-Reciprocal Plot
Enzyme Inhibition
Competitive inhibitor binds to the active site of the enzyme

Uncompetitive inhibitor binds to the enzyme-substrate complex, not free enzyme,


affects catalytic activity

A noncompetitive / mixed inhibitor binds both the enzyme and the enzyme-
substrate complex, alters both catalytic activity and substrate binding
Enzyme Inhibition
Competitive inhibition
Enzyme Inhibition
Competitive inhibition

Alcohol dehydrogenase
(liver)

Antifreeze (ehtylene glycol) poisoning in pets has similar principle


Enzyme Inhibition
Enzyme Inhibition
Uncompetitive inhibition: The inhibitor binds to the enzyme-substrate complex
Enzyme Inhibition
Noncompetitive / Mixed inhibition: The inhibitor binds to the enzyme and
enzyme-substrate complex
Metabolism
Catabolism
Anabolism

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