Enzymes (Part 2)

2009 W. H. Freeman and Company

Michaelis-Menten Equation
The rate of formation of product is V0 = k2 [ES]

V0 =

k2 [Etot] [S] Km + [S]

When the enzyme is saturated, [ES]=[Etot], Vmax= k2[Etot], then

V0 =

Vmax [S] Km + [S]

Michaelis-Menten Kinetics
The equation describes the kinetic behavior of

enzymes with hyperbolic dependence of V0 on [S] The rule that Km = [S], when V0 = Vmax, holds for all enzymes that follow Interpreting Vmax and Km Specificity constant kcat/Km, rate constant for the conversion of E + S to E + P

Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction

Determination of Kinetic Parameters The double-reciprocal plot

V0 = Vmax [S] Km + [S]

Km [S] 1 = + Vmax [S] Vmax [S] V0

Km 1 1 = + Vmax [S] Vmax V0

Lineweaver-Burk plot

Identify Constraints and Assumptions

Simplest Model Mechanism: E + S ES E + P One reactant, one product, no inhibitors

Total enzyme concentration is constant

Mass balance equation for enzyme: ETot = [E] + [ES] It is also implicitly assumed that: STot = [S] + [ES] [S]

Steady state assumption:

d [ ES ] = rateof formationof ES rateof breakdownof ES = 0 dt

What is the observed rate

Rate of product formation:

v net =

dP = k[ES] dt

Two-substrate Reactions
Kinetic mechanism: the order of binding of substrates and release of products When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms Sequential mechanism Ping-Pong mechanism

Sequential Kinetic Mechanism

We cannot easily distinguish random from ordered Random mechanisms in equilibrium will give intersection point at y-axis Lines intersect

Common mechanisms for enzyme-catalyzed bisubstrate reactions

Ping-Pong Kinetic Mechanism

Lines are parallel

Enzyme Inhibition
Inhibitors are compounds that decrease enzymes activity
Irreversible inhibitors (inactivators) react with the enzyme - one inhibitor molecule can permanently shut off one enzyme molecule - they are often powerful toxins but also may be used as drugs Reversible inhibitors bind to, and can dissociate from the enzyme - they are often structural analogs of substrates or products - they are often used as drugs to slow down a specific enzyme

Reversible inhibitor can bind:

To the free enzyme and prevent the binding of the substrate To the enzyme-substrate complex and prevent the reaction

Competitive Inhibition
Lines intersect at the y-axis

Uncompetitive Inhibition
Lines are parallel

Mixed Inhibition
Lines intersect left from the y-axis

Classification of Reversible Inhibitors

Reversible inhibitor can bind: To the free enzyme and prevent the binding of the substrate To the enzyme-substrate complex and prevent the reaction

Irreversible Inhibitors
Bind covalently or destroy a functional group on the enzyme, that is essential for enzymes activity, or form a stable noncovalent interaction Useful in identifying amino acids with catalytic activity Mechanism-based inactivators

Regulatory Enzymes
Enzymes that have Multisubunit proteins greater effect on the Metabolic enzymes rate of the could be regulated by multireaction sequence binding or proteolytic Allosteric cleavage
(noncovalent) regulation Reversible covalent modification

Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators

Feedback inhibition

Threonine dehydratase (E1) is specifically inhibited allosterically by L-isoleucine, the end product of the sequence, but not by any of the four intermediates (A to D).

Substrate-activity curves for allosteric enzymes