Enzyme kinetics – Definition

• Study of rates of catalytic reactions involving substrates and enzymes

ENZYMATIC REACTION VARIABLES

Substrate (S)

• Substance that enzyme modifies
• Typically the controlled variable

Velocity (V)

• Rate of product formation; typically the measured variable

Maximum velocity (Vmax)

• Reached when every enzyme’s active site is bound by a substrate
• Determined experimentally (reactions do not typically reach Vmax)

Reaction constant (Km)

• Substrate concentration corresponding to 1/2 of maximum velocity
• Determined experimentally
• Determines binding affinity of enzyme to substrate

MICHAELIS-MENTEN MODEL

• Examines reaction of single substrate w/ single enzyme to create product.

Assumptions

• No intermediates
• No product inhibition
• No allostericity or cooperativity
• Total enzyme concentration is constant (and [S] >>> [E])
• Pseudo-steady-state hypothesis: rate of [ES] formation = rate of [ES] breakdown
• Initial velocity (rate measured as soon as substrate/enzyme are mixed)

First-order kinetics

• Change in [S] changes velocity of rxn

Zero-order kinetics

• Change in [S] does NOT change velocity of rxn

Vmax

• Asymptotic: rxn rate approaches Vmax, never actually reaches it

Km

• Estimates enzyme binding affinity
• High affinity = requires a low [S] to reach 1/2 Vmax

LINEWEAVER-BURK MODELS

• Uses inverse of variables in Michaelis-Menten model to represent graph as straight line

1/Vmax

• Y-intercept: x = zero.

1/Km

• X-intercept: y=0

FACTORS THAT AFFECT REACTION VELOCITY

• Increasing temperature: increases reaction velocity until denaturation
• Change in pH: can denature enzymes –> loss of function –> ionization of amino acids (changes active site conformation
• [E] and [S] substrate affect reaction velocity