ENZYMES: KINETICS experimentally; are not necessarily equal to the

Lectured by: Dr. Marion Rivera coefficient of the balanced equation although
frequently they are.
ENZYME KINETICS
- Concerned with the quantitative measurement ORDER OF A REACTION
of the rates of enzyme - catalyzed reactions and the Defined as the exponents in the rate
factors that affect these rates. equation.
Overall order of a reaction is the sum of all
KINETIC THEORY exponents.
For two molecules to react, they must: Reaction is first order with
Approach within bond-forming distance of Rate = k [A]1
respect to A
one another or collide Reaction is first order with
Must possess sufficient kinetic energy to respect to A; first order
overcome the energy barrier for reaching the Rate k [A]1 [B]1
with respect to B; second
transition state. order overall
Reaction is zero order; rate
Anything that increases the frequency and the is constant and does not
energy of collision between substrates will Rate = k [A]0
depend on concentration of
increase the rate of the reaction. reactant.

ENERGY BARRIER FOR CHEMICAL

REACTIONS A+ B P

Rate1 = k1 [A]n [B]m

Rate-1 = k-1 [P]

At equilibrium, rate of conversion of

substrates to product equals the rate at which
products are converted to substrates
Rate1 = Rate-1
n m
k1 [A] [B] = k-1 [P]
k1 = [P]
k-1 = [A]n [B]m
k1 = keq equilibtium constant
k-1

G = -RT in keq
FACTORS THAT AFFECT REACTION RATE
FACTORS THAT AFFECT RATES OF
1. TEMPERATURE
ENZYME-CATALYZED REACTIONS
Increase TEMPERATURE increase
KINETIC energy of molecules increase
1. TEMPERATURE
MOTION and FREQUENCY with which
Initially, reaction rate increases as
molecules COLLIDE.
temperature rises (due to increase kinetic energy
2. REACTANT CONCENTRATION
of reacting molecules)
Frequency with which molecules collide is
DIRECTLY PROPORTIONATE to their Eventually, further increase in
concentration. temperature breaks hydrogen and hydrophobic
bonds that maintains 2o and 3o structure
A+ B P denaturation loss of catalytic activity

Rate [A] [B] [P] EFFECTS OF TEMPERATURE

t t t

Negative sign in the concentrations of A and

B indicates that A and B are being used up
while P is being produced.

Rate ~ [A]n [B]m or Rate = k [A]n [B]m

* k is a proportionality constant called rate constant.

* Exponents n and m must be determined
 TEMPERATURE
o Enzymes from humans generally
exhibit stability at temperature up to 45 - 55 C
o Q10 or temperature coefficient
Factor by which the rate of a
biologic process increases for a 10oC increase in
temperature
o For most biologic processes, Q10 = 2;
rate doubles for a 10o C rise
3. SUBSTRATE CONCENTRATION
o Velocity of reaction increases as
concentration of substrate is increased (first order
kinetics), however there will come a point when
further increases in substrate will not increase
velocity (zero order)

1. pH
o Affects enzyme activity by:
Enzyme denaturation at low or
high pH
Alteration in charged state of
enzyme and/or substrate
Change in the charge of group
which is distal to region where substrate bound but
which is needed to maintain 3o and 4o structure

4. ENZYME CONCENTRATION
o Initial velocity of reaction is directly
proportional to concentration of enzyme, providing
substrate is in excess.

MICHAELIS-MENTEN APPROACH TO
- +
Enz + SH EnzSH ENZYME KINETICS

At low pH : Enz- + H+ EnzH E+S ES P+E

At high pH : SH+ S + H+
Initial velocity (Vi)
OPTIMUM pH OF SOME ENZYMES Velocity measured immediately after enzyme
and substrate are mixed and no product has been
ENZYME OPTIMUM pH converted to substrate.
Pepsin 1.5 Rate of formation of ES
Catalase 7.6 Vi = k1 [E] [S]

Trypsin 7.7 Rate of breakdown of ES

Fumarase 7.8 Vi = k-1 [ES] + k2 [ES]
= (k-1 + k2) [ES]
Ribonuclease 7.8
Arginase 9.7 At steady state:
Rate of formation of ES = Rate of breakdown of ES
k1 [E] [S] = (k-1 + k2) [ES]
[E] [S] = k-1 + k2 = Km Michaelis constant
[ES] k1

[ES] = [ET] [S] vi = k2 [ES]

Km + [S] At Vmax: ET = ES
Vmax = k2 [ET]
vi = k2 [ES]
vi = (k2) [ET] [S]
Km + [S] vi = Vmax [S]
Km + [S]
SIGNIFICANCE OF Km AND Vmax LINEWEAVER-BURK PLOT (DOUBLE
RECIPROCAL PLOT)
At low [S], initial velocity is proportional to
substrate concentration. Converts hyperbolic curve to a straight line
At high [S], initial velocity is maximal MICHAELIS-MENTEN EQUATION
When [S] = Km; vi= V max 1 = Km 1 + 1
vi Vmax [S] Vmax
2 y = m x + b
*When the substrate concentration is equal to
Km value, the initial velocity (vi) is half-maximal Equation for a straight line:
y = mx + b
m = slope
b = y intercept

Kd = k-1 ; Dissociation constant, Kd is the

k1 inverse of the affinity of the enzyme
for its substrate
Km can be considered an inverse measure of the
affinity of the enzyme for the substrate.
*The lower the Km, the higher is the affinity
e.g.
Enzyme Substrate Km (mM)
Hexokinase Glucose 0.15
Fructose 1.5

*Which substrate has a greater affinity for

hexokinase? Answer: Glucose

Vmax is related to the turnover number of an

enzyme, a quantity equal to the catalytic constant, EADIE-HOFTEE PLOT
kcat An alternative linear transformation of the
Vmax = turnover number = kcat Michaelis-Menten Equation is the Eadie-Hofstee
[ET] transformation:
Turnover number is the number of moles of vi = vi 1 + Vmax
substrate that react to form product per mole of [S] = Km Km
enzyme per unit time
HILL EQUATION
Describes behavior of enzymes that exhibit
cooperative binding of substrate
Analogous to binding of O2 by hemoglobin
Shape of curve is sigmoidal

log vi = n log [S] - log k

Vmax-vi

*When n=1, all binding sites behave independently NON-COMPETITIVE INHIBITOR

and simple Michaelis-Menten kinetics behavior is Bears no structural resemblance to substrate;
observed binds to a different site on the enzyme
*When n>1, enzyme exhibit positive cooperativity

KINETICS OF ENZYME INHIBITORS

COMPETITIVE INHIBITOR
Raises the apparent Km (Km) for the
ENZYME INHIBITORS substrate
At high substrate concentration, Vmax is
unchanged
NON-COMPETITIVE INHIBITOR
Do not affect Km; lowers Vmax
UNCOMPETITIVE INHIBITOR
Binds only to ES complexes at locations other
than the catalytic site.
Substrate binding modifies enzyme structure,
making inhibitor-binding site available.
Inhibition cannot be reversed by substrate.
Apparent Vmax decreased; Km is decreased.

COMPETITIVE INHIBITOR
Resembles the substrate; compete for the same
binding site on the enzyme

e.g.
Substrate Product Competitive
Inhibitor
Succinate Fumarate Malonate
 Employed for determining inhibition constant (Ki)
which indicates the potency of an inhibitor
*The lower the Ki, the more effective the
inhibitor

For a competitive inhibitor, the lines converge

above the x-axis, and the value of [I] where they
intersect is Ki

For a non-competitive inhibitor, the lines

converge on x-axis, and the value of [I] where
they intersect is Ki

IRREVERSIBLE ENZYME INHIBITOR

Forms a non-dissociable complex with the

enzyme
Cyanide is a classic example of an irreversible
enzyme inhibitor; inhibits cytochrome oxidase,
an enzyme in the respiratory chain
The kinetic effect of irreversible inhibitors is to
decrease the concentration of active enzyme, thus
decreasing the maximum possible concentrations
of ES complex. Dixon plot analysis of Morin, a plant flavonoid
that inhibits the enzyme amino acyl-tRNA
DRUGS AS ENZYME INHIBITORS synthetase

If the drug requirement is to increase the

intracellular concentration of the substrate, either
a competitive or non-competitive inhibitor will
serve, since both will inhibit the utilization of
substrate, so that it accumulates.
E
S P
If the drug requirement is to decrease the
intracellular concentration of the substrate, then
the inhibitor must be non-competitive.
As unused substrate accumulates, the increasing
substrate concentration overcomes the effect of
the competitive inhibitor. Increasing the *What type of enzyme inhibitor is this plant
concentration of substrate does not affect a flavonoid? Answer: Competitive
non-competitive inhibitor.
BISUBSTRATE REACTIONS
DIXON PLOT
Enzymatic reactions involving two substrates and
Plot of 1/vi against concentration of inhibitor at yielding two products (Bi-Bi reaction)
different fixed concentration of substrate Account for ~60% of known biochemical
 reactions

Are either:
TRANSFERASE reactions in which the enzyme
catalyzes the transfer of a specific functional
group X from one of the substrate to the other
OXIDATION-REDUCTION reactions in which
reducing equivalents are transferred between two
substrate
Examples of Bi-substrate reactions:

1. SEQUENTIAL REACTIONS
- Reactions in which all substrates must
combine with the enzyme before a reaction
can occur and products be released
- Also called single displace reaction because
group undergoing transfer is usually passed
directly, in a single stem, from one substrate
to the other
- Types:
a.) Ordered- compulsory order of substrate
addition to the enzyme
b.) Random- no preference for order of
substrate addition
2. PING-PONG REACTIONS
- One or more products are released before all
substrate have been added
- Enzyme alternates between two forms E
and F
- Also called double displacement reactions

EXAMPLE OF PING-PONG REACTION: