TPN 202 BASIC FOOD CHEMISTRY AND

BIOCHEMISTRY
3 (1-2)

Coordinator:
Dr. Dian Herawati

Food Technology Study Program

Department of Food Science and Technology
IPB University

http://fst.ipb.ac.id
Internationally Accredited Study Program by IFT and IUFoST

TPN 202

Enzyme Kinetics

Food Technology Study Program

Department of Food Science and Technology
Course objectives

After this unit, students are expected to be able to:

• Explain the principle of enzyme kinetics
• Explain how to measure enzyme kinetics
Introduction
❖ Enzyme kinetics: determine the rate of the reaction and how
it changes in response to changes in experimental parameters
❖ This is the oldest approach to understanding enzyme
mechanisms and remains the most important

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Enzyme kinetics
❖ A key factor affecting the rate of a reaction
catalyzed by an enzyme is the concentration of
substrate, [S].
❖ A plot of V (velocity) vs [S] is often hyperbolic
The explanation of V vs [S] plot
1. As [S] is first increased, the initial
rate or velocity (V0) increases with
increasing substrate concentration
➔ V is proportional to [S]
2. As [S] increases, V increases less
and less ➔ V is NOT proportional
to [S] in this range
3. Finally, V doesn’t increase anymore
and velocity reaches its maximum
(Vmax) ➔ Enzyme is working as
fast as it can
4. Velocity won’t change no matter
how much substrate is present. At
this point, the enzyme is saturated
with substrate, S.
Michaelis and Menten theory (1913)
❖ The ES complex is the key to understanding enzyme
kinetic behavior
❖ The enzyme first combines reversibly with substrate to
form an enzyme-substrate complex in a relatively fast
reversible step

❖ The ES complex then breaks down in a slower second step

to yield the free enzyme and the reaction product P
 Michaelis and Menten theory (1913)
❖ In summary, the enzymatic reaction can be illustrated by this equation:

k1, k-1, and k2 are rate

constant for each step

❖ To derive the equation, they made 2 assumptions:

1. The reverse reaction (P → S) is not considered because the equation
describes initial rates when [P] is near zero
2. The ES complex is a STEADY STATE INTERMEDIATE, i.e. the concentration
of ES remains relatively constant because it is produced and broken
down at the same rate
Michaelis-Menten Equation

❖ V is the reaction rate (velocity) at a substrate concentration [S]

❖ Vmax is the maximum rate that can be observed in the reaction
❖ KM is the Michaelis constant
✓ KM is related to the affinity of the enzyme for the substrate
✓ units are in terms of concentration
✓ It is a combination of rate constants
 Michaelis constant (KM)
✓ Small KM means tight binding; large KM
means weak binding
✓ KM is also the substrate concentration at
which the enzyme operates at one half
of its maximum velocity
✓ Indicates how efficiently an enzyme
selects its substrate and converts to
product
✓ So, if an enzyme has a SMALL KM, then it
achieves maximal catalytic efficiency
(Vmax ) at a low substrate
concentration!
✓ KM is unique for each enzyme/substrate
pair
Michaelis constant (KM)

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 Linear transformation of Michaelis-Menten Equation

✓ The transformed equation is more useful in plotting experimental data

✓ One common transformation is derived simply by taking the reciprocal of both sides of
the Michaelis-Menten equation, or also called as Lineweaver-Burk equation
✓ The double-reciprocal plot of enzyme reaction rates is very useful in distinguishing
between certain types of enzymatic reaction mechanisms and in analyzing enzyme
inhibition

y= m x + b
 How to make M-M and Lineweaver-Burk curve

1) Amount of enzyme used ➔ E

2) Add substrate at several concentrations ➔ S (x )
3) Measure Product at Time t (P/t) ➔ vo (y )
4) Plot (x, y) hyperbolic curve, determine Vmax
5) If y = 1/2 Vmax , then [S] equals to Km

Vmax
1
vo vo
1/2
-1
Km 1
Vmax
Double reciprocal 1/S Km Direct plot S
 How toSubstrate
make M-M and Lineweaver-Burk
Product Velocity curve
Double reciprocal
[S] Absorbance v (mmole/min)
Data

no 1/S 1/v
1 0.25 0.21 → 0.42 4 2.08
2 0.50 0.36 → 0.72 2 1.56
3 1.0 0.40 → 0.80 1 1.35
4 2.0 0.46 → 0.92 0.5 1.16
(1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole
(2) Reaction time was 10 min

Double reciprocal
1.0 2.0
Direct plot

v 1/v 1.0
0.5 1.0
-3.8

0 0
0 1 [S] 2 -4 -2 0 2 4
1/[S]
Turnover number (Kcat, catalytic constant)
Definition:
✓ The limiting rate of an enzyme-catalyzed reaction at saturation
✓ Number of substrate molecules converted to product in a given unit of
time on a single enzyme molecule when the enzyme is saturated with
substrate
✓ How fast ES complex proceeds to E + P
✓ Number of catalytic cycles that each active site undergoes per unit time
✓ Rate constant of the reaction when enzyme is saturated with substrate
✓ First order rate constant (sec-1)
Turnover number = kcat = Vmax/[ET]
[ET] = total enzyme concentration
Catalytic efficiency
❖ The best way to compare the catalytic efficiencies of different enzymes
or the turnover of different substrates by the same enzyme is to
compare the ratio kcat/kM for the two reactions
❖ Catalytic efficiency (kcat/kM) Reflects both binding and catalytic events
and the best value to represent the enzyme’s overall ability to convert
substrate to product
❖ The diffusion controlled limit is 108 to 109 M-1s-1 ➔ maximum rate at
which two freely diffusion molecules can collide with each other in
aqueous solution (E and S)

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 Comparing catalytic efficiency of chymotripsin on different substrates

O R O

=
– –
H3C–C–N–C–C–O–CH3

–
H H
R= kcat / Km
Glycine –H 1.3 ╳ 10-1
Norvaline –CH2–CH2–CH3 3.6 ╳ 102

Norleucine –CH2–CH2–CH2–CH3 3.0 ╳ 103

Phenylalanine –CH2– 1.0 ╳ 105

(M-1 s-1)
 Enzyme inhibitor
I Competitive I Non-competitive I Uncompetitive
Substrate E
Cartoon Guide

S S E I
S

E S I I
I
Compete for S I
Inhibitor active site Different site

→ ES → E + P
E + S← E + S←→ ES → E + P → ES → E + P
E + S←
Equation and Description

+ + + +
I I I I
↓↑ ↓↑ ↓↑ ↓↑
EI EI + S →EIS EIS
[I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex
and competes with [S]; complex; Increasing [S] can only, increasing [S] favors
increasing [S] overcomes
not overcome [I] inhibition. the inhibition by [I].
Inhibition by [I].
 Enzyme inhibitor
I Competitive I Non-competitive I Uncompetitive
Vmax Vmax Vmax
vo vo
Direct Plots

Vmax’ Vmax’
I I I

Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM

Vmax unchanged Vmax decreased
Km increased Km unchanged Both Vmax & Km decreased
Double Reciprocal

1/vo I 1/vo I 1/vo

I
Two parallel
Intersect lines
at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax
at X axis

1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]

 Resume of enzyme kinetics Significance
Direct plot
kcat /Km Vmax [S] zero order
vo=
Km + [S] 1st order
kcat Observe vo change
E3
Turn over under various [S],
E2
number resulted plots
E1
yield Vmax and Km

k3 [Et] Vmax & Km

Competitive
Activity Unit Maximum Affinity with Double reciprocal
1 mmole velocity substrate

Inhibition
min
Non-competitive
Specific Activity Bi-substrate reaction also
unit Activity follows M-M equation, but
mg one of the substrate should
be saturated when estimate Uncompetitive
the other
 Why do we need to measure enzyme kinetics?
• To determine the chemical mechanism of an enzyme reaction, i.e., the
sequence of chemical steps that transform substrate into product
• To learn about the mechanism of enzyme inhibition and activation

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Thank you