Physical Biochemistry

Enzyme kinetics

Catalysis of chemical reactions

Catalysts are defined as species that increase the rate of a chemical reaction without altering the position of the equilibrium:
Equilibrium constant K is not affected.

Catalysts may directly participate in the reaction but are regenerated at the end of the reaction.
A+ C I I+ B J J Products + C
Catalyst

Overall reaction

A + B + C Products + C

Enzymes
Very high rate enhancements:
Typically 106 1014 compared to uncatalyzed rate. Diffusion-controlled/limited: Enhancement is so high, the rate essentially depends on fast a substrate can reach the active site.

High selectivity:
Only particular substrates can bind.

High specificity:
Only particular reaction is catalyzed:
E.g. Glucosyl transferase will only add glucose on to 2 position of another glucose, but not at the other positions 3,4 and 6.

Enzymes
Have ability to respond to external signals or their environment:
Activity of enzymes can be controlled or altered. Very important prerequisite to control metabolism.

Free energy of binding

Enzymes use the free energy of binding bG to change the free energy of activation G. Rate of reaction is determined by the free energy difference between transition state and reactants:
Reduction of this difference increases rate.

For single-substrate reactions, two ways:

Destabilize substrate / stabilize transition state. Altering the reaction mechanism.

Destabilize substrate / Stabilize transition state.

Binding usually favorable, but increases free energy of activation. Favorable free energy of binding for distorted substrates:
Distortion of substrate towards transition state. Lower free energy of activation.

Altering reaction mechanism

Enzyme provides functional groups that are more effective than the normal reactants:
Glu of enzyme acts as proton donor instead of water of solvent.

This changes the reaction mechanism towards a path with a lower free energy of activation:
Increases rate.

Free energy of binding is used to position the substrate in the best possible orientation with respect to the functional groups.

Single-substrate enzyme kinetics

Derivation of the Michaelis-Menten equation. Two ways:
Steady state approximation of enzymesubstrate complex ES. Equilibrium approximation:
ES in equilibrium with E and S slightly altered by the breakdown to products.
k E + S ES 2 E + Products k 1 k1

Steady-state approximation for ES

k E + S ES 2 E + Products k 1 k1

Steady-state approximation: Free enzyme concentration:

d [ ES] = k1 [ E ] [ S] k 1 [ ES] k 2 [ ES] = 0 dt

[ E ] = [ E ] total [ ES]

Concentration of enzyme present at the start of the reaction.

d [ ES ] = k1 ( [ E ] total [ ES ] ) [ S ] k 1 [ ES ] k 2 [ ES ] = 0 dt

Steady-state approximation for ES

d [ ES ] = k1 ( [ E ] total [ ES ] ) [ S ] k 1 [ ES ] k 2 [ ES ] = 0 dt
v = k 2 [ ES ] = k 2 [ E ] total [ S ] k 1 + k2 + [S] k1

[ E] total [S] [ ES] = k + k 1 2 + [ S]

k1
Km = k 1 + k2 k1

Vmax = k 2 [ E ] total

Vmax [ S] v= K m + [ S]

Rate of the reaction:

Michaelis-Menten equation.

Michaelis-Menten equation
Relates the rate v of the enzymecatalyzed reaction to the concentration of the free substrate [S]. Approximation when [S]total >> [ES]:

[ S ] = [ S ] total [ ES ] [ S ] total

Vmax [ S] v= K m + [ S]

Equilibrium approximation
k E + S ES 2 E + Products k 1 k1

Dissociation constant: K m =

[ E] [S] [ ES] = [ E] [S] [ ES] Km

= K m + [ S]

Define:

[ ES] = [ ES] = Km [ E] total [ E] + [ ES] [ E] + [ E] [S]

Km

[ E] [S]

[S]

Fraction of enzyme that 0 1 is active or has a bound reacting substrate.

Divide by [E], multiply by Km

Equilibrium approximation
=
K m + [ S]

[S]

0 1
=

Rate of the reaction:

Vmax [ S] v = Vmax = K m + [ S]
Michaelis-Menten equation

[ ES] [ E ] total

Vmax is the limiting (maximum) rate attainable, when all enzyme is present as ES.

Michaelis-Menten equation
[S] > >
Km

[S] >> Km:

Km + [S] [S] v = Vmax = k2 [ES]

[S] < < Km:

[S] < <

Km

Km + [S] Km v =Vmax [S] / Km Straight line with slope Vmax / Km

Vmax [ S] v= K m + [ S]

[S] = Km:
v = Vmax / 2.

Michaelis-Menten equation
Vmax = k 2 [ E ] total
Vmax is the limiting velocity of the reaction at a given concentration of the enzyme:
Enzyme fully saturated with substrate S.
k 1 + k2 Steady state : K m = k1 k 1 Equilibrium : K m = k1

Km is the Michaelis constant:

Steady state: Measure of life time of ES complex. Equilibrium approximation: Km is a dissociation constant.

Michaelis-Menten equation
kcat Vmax = [ E ] total

kcat is catalytic constant:

First order rate constant of the decomposition reaction into products. Same as k2 for single step decomposition. Also rate constant at high concentration of S: v = kcat [E]total.

Vmax = k 2 [ E ] total
k E + S ES 2 E + Products k 1 k1

Michaelis-Menten equation
kcat kA = Km
kA is specificity constant or catalytic efficiency:
Measures degree of selectivity. Maximum value is diffusion rate constant, when diffusion is the slowest step. Also second order rate constant at small concentration of S

v=

Vmax [ S] k [ E ] [ S] cat total = k A [ E ] total [ S] K m + [ S] Km

[S] < <

Km

Michaelis-Menten equation
Turnover number:
Moles of substrate consumed or product formed per mole enzyme per unit time. Same as kcat.

Analysis of kinetic data

Fit experimental data to Michael-Menten equation with non-linear regression. Rearrange rate to obtain a straight line.
1 Km 1 1 = + v Vmax [ S] Vmax

v=

Vmax [ S] K m + [ S]

1 1 versus [S] v

Lineweaver-Burk plot: x-intercept: Km-1 Y-intercept (x=0): Vmax-1

v Vmax v = [ S ] Km Km

Eadie-Hofstee plot: v versus v Slope: -Km-1 [S] X-intercept (y=0): V

max

[S] = [S]
v

Vmax

K + m Vmax

[ ]

Hanes plot: S versus S X-intercepts (y=0): -Km v Slope: Vmax-1

[ ]

Worked example
The activity of the enzyme urease, which catalyses the reaction CO(NH2)2 + H2O CO2 + 2NH3 was studied as a function of urea concentration with the following results. What are the values of Km and Vmax.
[Urea] mM v mmol urea consumed (mg enzyme)-1 min-1 30 3.37 60 100 150 8.94 250 400

5.53 7.42

10.70 12.04

Microsoft Excel Worksheet

Limitations
Pre-steady-state period:
Reaction reaches not immediately equilibrium:
Can be used to obtain better information about reaction.

Rate equations much more complex.

Approach to equilibrium:
Assumption: Forward reaction towards products is irreversible. Practise: Reversible reaction if product concentration is high enough.

Product inhibition:
Product has an ability to bind to active site to become an competitive inhibitor.

Substrate inhibition:
Some enzymes are inhibited by high substrate concentration. Usually indicates a second binding site for substrate.

Enzymes with more than one active site:

Active sites may affect each other. Positive, negative cooperativity.

Temperature affect kinetics

Reaction usually follows Arrhenius equation for Tdependency. Enzyme:
Unfolds above melting temperature rendering it less active. Multistep reaction:
Different steps may be rate determining at different temperatures.

k = Ae

Ea RT

G = H T S

Temperature affect kinetics

Enzyme:
May exists in more than one active interconvertible conformation. Each active form has different free energy of activation.

Effects of pH
Changes in pH affects rates of enzymecatalyzed reactions by:
H+ and/or OH- appear in rate equation(s). Changes in ionization state of substrate:
Additional acid-base catalysis. Changes in substrate binding free energy:
Affects Km.

pH affect stability of enzyme:

Changes in ionization state of titrating residues:
May affect binding and therefore Km. May Vmax if titrating residues directly participate in reaction.

One titrating residue

Example:
Active site containing one titrating site. Enzyme is active when this titrating site is deprotonated. Enzyme is activated when pH is increased H+ acts as non-competitive inhibitor.

EH + : S E : S + H +
Inactive Active

Km

One titrating residue

Typical plots Vmax is higher at higher pH.
1.5 Occupancy Charge state 1

0.5

0
0 1 2 3 3.6 3.8 4 4.2 4.4 5 6 8 10 12 14

-0.5

-1

-1.5 pH

Two titrating sites

Active site contains two titrating residues:
One with low pKa1. One with high pKa2.

HX E YH + HX E Y + X E Y
+H +H

H+

H+

Active Inactive

pK a1 < pK a 2

Inactive

Two titrating sites

Typical plot for well separated pKas. Maximum narrows when pKa are overlapping.

Two titrating sites

Effective Vmax may not reach ideal Vmax, when pKas are too close.

HX E YH + HX E Y + X E Y
+H +H

H+

H+