Enzymes

Kinetics, Inhibtors

Known conditions needed for experimmental assays

Buffer, substrate conc., temp, enzyme conc.

ES association constant

K_A = [ES] / [E][S] | (k₁/k_-1)

ES dissociation complex

K_D = 1/K_A = [E][S] / [ES] | (k_-1/k₁)

V₀ (Initial Velocity)

Earliest speed of k₂

V_max

When [S] is saturated (high [S]) —> =V₀

Michaelis complex

ES

Steady state (Michaelis Menten)

When [ES] and [E] do not change

Transient Phase (Michaelis Menten)

Very fast step before steady state

Michaelis Constant

K_{M :} K_M= [S] at ½ of V_max

K_M

Estimate of substrate binding affinity

Michaelis-Menten Equation

Specificity constant

k_cat/K_M

Specificity constant definition

Measure of substrate preference/catalytic efficiency

M^-1s^-1

Specificity constant units

Larger value for great enzyme relative to poor enzyme

Specificity constant trend

Catalytic Proficiency

Measure of an enzyme’s affinity for transition state

Lineweaver-Burke

Double reciprocal plot

Slope (m) : K_M/V_max

Y-intercept (b) : 1/V_max

X-intercept: -1/K_M

Lineweaver-Burke Plot parameters

Irreversible inhibition

Enzyme permanently destroyed (covalent)

Reversible inhibition

Enzyme not destroyed-can be reactivated upon removal of inhibitor

Competitive, uncompetitive, non-competitive

3 forms of reversible inhibitors

Penicillin

Irreversible inhibitor example

Competitive inhibition

Reversible inhibition- substrate and inhibitor are mutually exclusive, transition state substrate and inhibitor are similar structures

a (alpha)

The measure of the effectiveness of I

a = 1 + ([I]/K_I)

a (alpha) equation

K_I

Enzyme inhibitor dissociation constant

K_I equation

K_I=([E][I])/[EI]

K_M^app = (a)(K_M) ; K_M^app- increase, V_max-unchanged

Effect of competitive inhibition on apparent K_M^app

Competitive inhibition graphs

Competitive inhibition degree

Uncompetitive inhibition

Reversible inhibition- inhibitor only bonds to ES complex

K_I’

ES complex inhibitor dissociation constant

K_I’ equation

K_I’=([E][I])/[EI]

K_M^app and V_max^app lowered by a"‘

Effect of uncompetitive inhibition on apparent K_M^app

Uncompetitive inhibition graphs

Non-competitive inhibition

Reversible inhibition- inhibitor bonds both the free enzyme and ES complex; inhibitor binds at different site than the active site

Pure non-competitive inhibitors

Mixed non-competitive inhibitors

Non-competitive inhibition graphs

Key concepts