ENZYME KINETICS

CHE 142 – Biochemical Engineering

Basic Reaction Theory
 Reaction rate
Consider the irreversible reaction:

aA + bB → yY + zZ

Total rate: Volumetric rate: Specific rate:

−dM A RA −dC A ⎛ 1 1 ⎞ dM A
RA = rA = = rA = − ⎜ or ⎟
dt V dt ⎝ E X ⎠ dt

CHE 142 Biochemical Engineering

 Reaction rate
Specific rate – provides a direct reflection of cataylst peformance

⎛ 1 1 ⎞ dM A
rA = − ⎜ or ⎟
⎝ E X ⎠ dt
unit of enzyme activity – amount that catalyzes the conversion of 1 µmole
of substrate per minute at the optimal T, pH and substrate concentration

In terms of units of enzyme activity:

⎛ 1 1 ⎞ dCA
rA = − ⎜ or ⎟
⎝ e x ⎠ dt
where e and x are enzyme and cell concentrations, respectively.
CHE 142 Biochemical Engineering
 Reaction kinetics

Reaction kinetics – refers to the relationship

between the rate of reaction and the
conditions that affect it
q  reactant concentration

q  temperature

CHE 142 Biochemical Engineering

 Reaction kinetics
Consider the irreversible reaction:

aA + bB → yY + zZ

The volumetric rate of the reaction can be expressed as:

α
rA = kC C A
β
B

where:
k – reaction rate constant or rate coefficient
CA, CB – concentrations of reactants A and B
α,β – reaction order
CHE 142 Biochemical Engineering
 Effect of temperature on
reaction rate
The variation of the rate constant with T is described by the Arrhenius equation:

− E / RT
k = Ae
E
ln k = ln A −
RT
where:
k – reaction rate constant or rate coefficient
A – Arrhenius constant
E – activation energy for the reaction
T – absolute temperature

CHE 142 Biochemical Engineering

General Reaction Kinetics for
Biological Systems
 Reaction order
v Zero-order kinetics
v First-order kinetics
v  Second-order kinetics
v Michaelis-Menten kinetics
 Zero-order kinetics
The reaction rate of independent of reactant concentration:

A → Products

rA = k0
In a closed constant V system:
dCA
− = k0
dt
CA = CA0 − k0t
where:
k0 – zero-order reaction rate constant (M/s)
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r
 Zero-order kinetics
Dependence of k0 on catalyst concentration:

' "
k0 = k e 0 k0 = k x 0

where:
k0’– specific zero-order reaction rate constant
e – enzyme concentration
x – cell concentration

CHE 142 Biochemical Engineering

 Zero-order kinetics
Example:
Time (min) O2 Conc.
Serratia marcescens is cultured (mmol/L)
in minimal medium in a small 0 0.25
stirred fermenter. Oxygen
consumption is measured at a 2 0.23
cell concentration of 22.7 g/L
dry weight. 5 0.21

a. Determine the rate constant 8 0.20

for oxygen uptake.
b. If the cell concentration is 10 0.18
reduced to 12 g/L, what is the
value of the rate constant? 12 0.16

15 0.15

CHE 142 Biochemical Engineering

 First-order kinetics

A → Products

rA = k1CA
In a closed constant V system:

dCA
− = k1CA
dt
− k1t
ln CA = ln CA0 − k1t CA = CA0 e
where:
k1 – first-order reaction rate constant (1/s)
CHE 142 Biochemical Engineering
 First-order kinetics
Example:
Time (day) Petroleum HC
Soil contaminated with crude Conc. (mg/kg)
oil is treated using a mixed 0 1375
population of indigenous
bacteria and fungi. The 7 802
concentration of total
14 695
petroleum hydrocarbons in soil
sample is measured as a 21 588
function of time over a 6-week
period. 28 417
a. Determine the rate constant 35 356
for petroleum degradation.
b. Estimate the contaminant 42 275
concentration after 16 days.

CHE 142 Biochemical Engineering

 Reversible first-order reactions
Most biochemical reactions are reversible and equilibrium does not lie far to one side:

k1
A ←# →P

Rate equation:
d[A]
rA = − = k1CA − k−1CP
dt
At equilibrium:

CP e k1
0 = −k1CAe + k−1CPe K eq = =
CAe k−1
where: k1 – first-order, forward reaction rate constant
k-1 – first-order, backward reaction rate constant
CHE 142 Biochemical Engineering
Second-order reactions:
Second-order reactions:
 Remember:

v The order of a reaction must be determined

experimentally.
v Understanding the stoichiometry of a reaction is
insufficient to predict the rate law of the reaction.
Michaelis-Menten Kinetics
 Rate expression for ezyme-catalyzed
reactions

•  Michaelis-Menten approach – rapid equilibrium

assumption
•  Briggs and Haldane approach – quasi-steady-state
assumption

CHE 142 Biochemical Engineering

 Michaelis-Menten kinetics

•  Kinetics of simple enzyme-catalyzed reactions are often

referred to as Michaelis-Menten kinetics or saturation
kinetics.
•  These models are based on data from batch reactors
with constant liquid volume in which the initial
substrate, [So], and enzyme, [Eo], concentrations are
known.

CHE 142 Biochemical Engineering

Michaelis-Menten kinetics

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 Michaelis-Menten kinetics

The qualitative features of

enzyme kinetics are similar
to Langmuir-Hinselwood
kinetics.

turnover number
(catalytic constant)

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 Michaelis-Menten kinetics

vmax = kcat ea
vmax
kcat =
E0
ea, E0– concentration of active enzyme
kcat – catalytic constant
– number of S molecules converted to P per enzyme per unit time
CHE 142 Biochemical Engineering
 Turnover numbers

Enzyme kcat (s-1)

Carbonic anhydrase 1,000,000

Acetylcholinesterase 25,000

Lactate dehydrogenase 1,000

DNA polymerase I 15

Lysozyme 0.5

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 Michaelis-Menten kinetics

Catalytic efficiency

Km – substrate concentration at which v = vmax/2, that is, half of

the enzyme’s active sites are saturated with substrate
– relative measure of the substrate binding affinity
– lower values imply higher enzyme affinity for the substrate

Catalytic efficiency = kcat / Km

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 Turnover numbers

Enzyme Substrate KM (µM)

Carbonic CO2 8000

anhydrase
Lysozyme Hexa-NAG 6

Penicillinase Penicillin 50

Arg t-RNA Arginine 3

synthetase
t-RNA 0.4

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 Mechanistic models for simple
enzyme kinetics

where v is the rate of product formation or substrate consumption in moles/l-s.

v  The rate of variation of the ES complex is

(3.3)

v  Since the enzyme is not consumed, the conservation equation oh the
enzyme yields
 The rapid equilibrium assumption
v  Assuming a rapid equilibrium between the enzyme and substrate to
form an [ES] complex, we can use the equilibrium coefficient to
express [ES] in terms of[S].
v  The equilibrium constant is

v  Since [E] = [Eo] - [ES] if enzyme is conserved, then

 The rapid equilibrium assumption
where Km’ = k-1/k1, often called the Michaelis-Menten constant,
which is the dissociation constant of the ES complex.

where Vm = k2[E0].
 The quasi-steady-state assumption
v By applying the quasi-steady-state assumption

v Applying the enzyme conservation yields

 The quasi-steady-state assumption
v Solving for [ES],

v Substituting into the rate equation:

where Km is (k-1+k2)/k1 and Vm is k2[E0].

The quasi-steady-state assumption
Michaelis-Menten kinetics
Michaelis-Menten kinetics

Zero-order region

1st-order region
 Michaelis-Menten kinetics

For the zero-order region, [S] >> Km

vmax [S]
v≈
[S]
For the 1st-order region, [S] << Km

vmax [S]
v≈
Km
 Experimentally determining rate parameters
for Michael-Menten type kinetics

v The determination of values for Km and Vm with high precision

can be difficult.
v Typically, experimental data are obtained from initial-rate
experiments.
v A batch reactor is charged with a known amount of substrate
[S0] and enzyme [E0].
v The product or substrate concentration is plotted against time.
v = d[P] / dt t=0 = −d[S] / dt t=0
v This value of v then depends on the value of [E0] and [S0]
charged to the reactor.
Michaelis-Menten plot

Accurate determination
of Vm is difficult.
 Double-reciprocal plot

v A double-reciprocal plot gives good estimates on Vm, but not

necessarily on Km.
 Double-reciprocal plot

.
Eadie-Hofstee plot
 Hanes-Woolf plot

v This plot is used to determine Vm more accurately.

 Batch kinetics
v The time course of variation of [S] in a batch enzymatic
reaction can be determined from

d[S] Vm [S]
v=− =
dt K m +[S]
v by integration to yield

[S0 ]−[S] K [S0 ]

Vm − = ln
t t [S]
 Interpretation of Km and Vm
v Vm is a function of the rate parameter k2 and the initial enzyme
level, [E0].
v As [E0] changes, so does Vm.

v  A unit is the amount of enzyme that gives a predetermined

amount of catalytic activity under specific conditions.

v The specific activity is the number of units of activity per

amount of total protein.

v The enzyme may be denatured if it unfolds or has its three-

dimensional shape altered by pH extremes or temperature
during purification.
 Example 3.2
The following data have been obtained for two different initial
enzyme concentration for an enzyme-catalyzed reaction.
a. Find Km. v ([E0] = 0.015 g/L) [S] v ([E0] = 0.00875 g/
(g/L-min) (g/L) L) (g/L-min)
b. Find Vm for
1.14 20.0 0.67
[E0] = 0.015 g/L.
0.87 10.0 0.51
c. Find Vm for
0.70 6.7 0.41
[E0] = 0.00875 g/L.
0.59 5.0 0.34
d. Find k2.
0.50 4.0 0.29
.
0.44 3.3
0.39 2.9
0.35 2.5
Enzymes kinetics

END