SCE 3204 Biochemical

engineering
By
Kissa R. Alunga
BIC, Ms Chem eng, CBRNe Specialist

Lecture 3
Michaelis-Menten model & effects of substrate
concentration

Michaelis-Menten
Model:
“According to this model the
enzyme reversibly combines
with substrate to form an ES
complex that subsequently
yields product, regenerating
the free enzyme.”
Michaelis-Menten Equation (Derive this equation???)
• “It is an equation which describes how reaction
velocity (rate of reaction) varies with substrate
concentration.”
Michealis-Menten Analysis
 Our starting point is that the catalytic rate is equal
to the product of the concentration of the ES
complex and k2.
V0  k 2 ES  ( 1)

• The rates of formation and breakdown of ES are given by:

d ES 
Rates of formation  k1 E S  (2)
dt

Rates of breakdown  d ES   k  k ES  (3)

1 2
dt
6 CHE324 Introduction to Biochemical Engineering
 Assuming steady-state condition

k1 E S   k 1  k 2 ES  (4)

By rearranging equation, we obtain
E S   k 1  k 2   K (5)
ES  k1
M

• KM, called the Michaelis constant; KM has the units of

concentration. KM is an important characteristic of
enzyme-substrate interactions and is independent of
enzyme and substrate concentrations
ES   E S 
(6)
KM
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 Examining eqn 6
The concentration of uncombined substrate [S] is very
nearly equal to the total substrate concentration. The
concentration of uncombined enzyme [E] is equal to
the total enzyme concentration [E]T minus the
concentration of the ES complex.
E   E T  ES  (7)
Substituting this expression for [E] in equation 6
gives
ES   E T  ES  S 
(8)
KM

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 Solving equation 8 for [ES] gives

ES   E T S  (9)
S  K M
• By substituting this expression for [ES] into equation 1, we obtain

V0  k 2 E T
S  (10)
S  K M
• The maximal rate, Vmax, is attained when the catalytic sites on the
enzyme are saturated with substrate—that is, when [ES] = [E] T. Thus,
eq.1 becomes;
Vmax  k 2 E T (11)
• Substituting eq. 11 into eq. 10 yields the Michaelis-Menten equation:

V0  Vmax
S  (12)
S  K M
9 CHE324 Introduction to Biochemical Engineering
Michealis-Menten Analysis
 The Michaelis constant, KM, has two meanings.
1. KM is the concentration of substrate at which
half the active sites are filled.
2. Thus, KM provides a measure of the substrate
concentration required for significant
catalysis to occur.
3. When the KM is known, the fraction of sites
filled, fES, at any substrate concentration can
be calculatedf from

V

S
K M  S 
ES
Vmav
11 CHE324 Introduction to Biochemical Engineering
 Eq. 12 accounts for the kinetic data.
1. At very low substrate concentration, when [S] is
much less than KM, V0 = (Vmax/KM)[S]; that is, the
rate is directly proportional to the substrate
concentration. The reaction is now first order in
substrate.
2. At high substrate concentration, when [S] is much
greater than KM, V0 = Vmax; that is, the rate is
maximal, independent of substrate concentration,
thus the reaction is zero-order in substrate,
implying most of the enzyme is found in the
bound state, [ES]
12 CHE324 Introduction to Biochemical Engineering
  KM is related to the rate constants of the individual steps in the

catalytic scheme.
 KM is defined as (k-1 + k2)/k1.
 Consider a limiting case in which k-1 is much greater than k2.
Under such
circumstances, the ES complex dissociates to E and S much more
rapidly than product is formed.kUnder these conditions (k-1>>k2),
KM  1
k1

• When this condition is met, KM is a measure of the

strength of the ES complex: a high KM indicates weak
binding; a low KM indicates strong binding.

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 A Physiological Effect
The physiological consequence of
KM is illustrated by the sensitivity
of some individuals to ethanol.
Such persons exhibit facial
flushing (rapid reddening of skin)
and rapid heart rate after ingesting
even small amounts of alcohol. In
the liver, alcohol dehydrogenase
converts ethanol into acetaldehyde.
Alcohol
CH 3CH 2OH  NAD    CH 3CHO  H   NADH
 dehydrogenase

14
 A Physiological Effect
The acetaldehyde is the
cause of the symptoms
when present at high
concentrations, it is
processed further to acetate
by acetaldehyde
dehydrogenase.
Acetaldehyde
CH 3CHO  NAD     CH 3COO  2 H   NADH
dehydrogenase

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 A Physiological Effect
Most people have two forms of the acetaldehyde
dehydrogenase, a low KM mitochondrial form and
a high KM cytosolic form.
In susceptible persons, the mitochondrial enzyme
is less active and acetaldehyde is processed only
by the cytosolic enzyme. Because this enzyme
has a high KM, less acetaldehyde is converted into
acetate; excess acetaldehyde escapes into the
blood and accounts for the physiological effects.
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 The Significance of KM

The KM values of enzymes range widely. For most

enzymes, KM lies between 10-1 and 10-7 M. The KM value
for an enzyme depends on the particular substrate and on
environmental conditions such as pH, temperature, and
ionic strength
Enzyme Substrate KM(μM)
Pyruvate carboxylase Pyruvate 400
HCO3- 1000
ATP 60
Arginine-tRNAsynthetase Arginine 3
tRNA 0.4
ATP 300
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 The Significance Vmax Values
The maximal rate, Vmax, reveals the turnover
number of an enzyme (kcat or k2) which is the
number of substrate molecules converted into
product by an enzyme molecule in a unit time
when the enzyme is fully saturated with
substrate. The maximal rate, Vmax, reveals the
k 2 E T if the
Vmaxanenzyme
turnover number of
concentration of active Vsites [E] T is known.
k 2 max
E T
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 Enzyme Turnover number (s-1)
Carbonic anhydrase 600,000
Chymotrypsin 100

Example, which means that each carbonic anhydrase

molecule can produce up to 600,000 (bicarbonate
ions) molecules of product per second. This turnover
number is one of the largest known.

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 Catalytic efficiency
It’s the measure of how efficiently an enzyme
converts a substrate into product. It’s evaluated
by;
k cat k2 k1k 2
 
KM KM k 1  k 2

• It has a theoretical upper limit of 108-1010 (M-

s ); enzymes working close to this, such as
1 -1

fumarase, are termed superefficient.

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 Experimentally determined parameters for enzymes
Enzyme KM (M) kcat (1/s) kcat/KM (1/M.s)
Chymotrypsin 1.5 × 10−2 0.14 9.3
Pepsin 3.0 × 10−4 0.50 1.7 × 103
Tyrosyl-tRNA synthetase 9.0 × 10−4 7.6 8.4 × 103
Ribonuclease 7.9 × 10−3 7.9 × 102 1.0 × 105
Carbonic anhydrase 2.6 × 10−2 4.0 × 105 1.5 × 107
Fumarase 5.0 × 10−6 8.0 × 102 1.6 × 108

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 Linear plots of the Michaelis – Menten equation

Determining Vmax and KM from experimental data

can be difficult because the curve approaches the
Vmax value asymptotically and the most common
way is to determine initial rates, V0, from
experimental values of [P] or [S] as a function of
time.
• Line that a graph
approaches but
never touches

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 Hyperbolic graphs of V0 vs. [S] can be
transformed into the following linear
equations;
• Lineweaver–Burk plot.
• Eadie–Hofstee diagram.
• Hanes–Woolf plot.

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 Lineweaver–Burk plot is a graphical
representation of the Lineweaver–Burk equation
of enzyme kinetics, described by Hans
Lineweaver and Dean Burk in 1934.

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 Derivation
The plot provides a useful graphical method for
analysis of the Michaelis-Menten equation.
Vmav S 
V0 
K M  S 

This is produced by taking the reciprocal of both sides

of the Michaelis–Menten equation to obtain the
double reciprocal curve.
1 K M  S  K M  1  1
    
V Vmav S  Vmav  S   Vmav
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 Eadie–Hofstee diagram
The Eadie–Hofstee plot is a graphical representation of
enzyme kinetics in which reaction rate is plotted as a
function of the ratio between rate and substrate
concentration and can be derived from the Michaelis-
Menten equation by inverting and multiplying
with Vmax:
V0
V0   K M  Vmax • Derive…. ?
S 

• A plot of V0 vs V0 /[S] will yield Vmax as the y-intercept,

Vmax /KM as the x-intercept, and KM as the negative slope.

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 Exercise-hand in after class
 The reaction between nicotineamide mononucleotide and ATP to
form nicotineamide-adenine dinucleotide and pyrophosphate is
catalyzed by the enzyme nicotinamide mononucleotide
adenylyltransferase.
 The following table provides typical data obtained at a pH of 4.95.
The substrate, S, is nicotinamide mononucleotide and the initial
rate, V, is the μmol of nicotinamide–adenine dinucleotide
[S] (mM) formed
V (μmol)
in a 3-min reaction period.
0.138 0.148
0.220 0.171
0.291 0.234
0.560 0.324
Determine values for Vmax and KM 0.766 0.390
27 CHE324 Introduction to Biochemical Engineering 1.460 0.493
THANK YOU

Any Questions?