CHAPTER 1:

ENZYME KINETICS AND

APPLICATIONS
(Part I : Kinetics of Enzyme Catalyzed
Reactions)

ERT 317 Biochemical Engineering

Sem 1, 2015/2016
Role of Bioprocess Engineering
exploit advances in biology to create new products
design biochemical processes & operate plants
develop energy resources
Develop new, environmentally friendly, and safer
processes to make the biochemical products that
people depend on.
Work in research and development laboratories,
creating polymeric materials with improved
performance and durability.
Work in manufacturing, making vaccines and antibiotics.
Invent new ways to keep our food and water supplies
safe.
Bioprocess Engineers Task
Minimize production of unwanted
byproducts
Separate the good (product) from the bad
(byproducts)
Recover the unused reactants
Maximize profit, minimize energy
consumption
Minimize impact on the environment
OPPORTUNITIES FOR BIOPROCESS
ENGINEERS

pharmaceuticals
polymers
energy
food
consumer products
biotechnology
electronic and optical materials.
OUTLINE
Introduction
Enzyme Structure
Enzyme Function
Enzyme Kinetics
A)Michaelis Menten Kinetics
B) The Rapid Equilibrium Assumption
C) The Quasi-Steady-State Assumption
Enzymes
There are many chemical compounds in the living cell.

How they are manufactured and combined at sufficient

reaction rates under relatively mild temperature and
pressure?

How does the cell select exactly which reactants will be

combined and which molecule will be decomposed?

Catalysis by ENZYME
Enzymes
Enzymes are biological catalysts that are protein molecules in
nature- react in mild condition

They are produced by living cells (animal, plant, and

microorganism) and are absolutely essential as catalysts in
biochemical reactions.

Almost every reaction in a cell requires the presence of a

specific enzyme related to its particular protein structure.

A major function of enzymes in a living system is to catalyze

the making and breaking of chemical bonds.

Therefore, like any other catalysts, they increase the rate of

reaction without themselves undergoing permanent chemical
changes.
Over 2000 enzymes have been identified

Often named by adding the - ase to the name of substrate

acted upon, or the reaction catalyzed such as urease, alcohol
dehydrogenase

The majority of cellular reactions are catalyzed by

enzymes
 Some protein enzyme required a non-protein group for their
activity.

Non protein group:

Cofactors: metal ions, Mg, Zn, Mn, Fe.
Coenzyme: complex organic molecule, NAD, FAD, CoA
Vitamins

Catalyze biochemical reactions

breaking, forming and rearranging bonds.

Catalytic function very specific and effective (Specific

because of conformational shape)
Dictated by the enzyme active site.
Some active sites allow for multiple substrates.
 Enzymes are catalysts
Catalyst: chemical that changes the rate of a
reaction without being consumed
Recycled (used multiple times)

Enzymes reduce the activation energy of a

reaction
Amount of energy that must be added to get a
reaction to proceed
Catalysts
A catalyst is unaltered during the course of a reaction and
functions in both the forward and reverse directions.

In a chemical reaction, a catalyst increases the rate at

which the reaction reaches equilibrium.

For a reaction to proceed from starting material to product,

the chemical transformations of bond-making and
bond-breaking require a minimal threshold amount of
energy, termed activation energy.

Generally, a catalyst serves to lower the activation

energy of a particular reaction.
Enzyme Function
Enzymes lower the activation energy of reaction
catalyzed
( They do this by binding to the substrate of the reaction,
and forming an enzyme-substrate (ES) complex)

Substrate binds to a specific site on the enzyme

called the active site

Multi-substrate reactions possible

Lock and key model

The activation energy for
the decomposition of
hydrogen peroxide varies
depending on the type of
catalysis.

Type of Activation
catalysis energy

Uncatalyzed 18
reaction at kcal/mol
20C

Enzymatically 7 kcal/mol
catalyzed
(catalase)

Chemically 13
catalysed (by kcal/mol
collodial Enzyme lower the activation energy of the reaction
platinum)
by binding the substrate and forming an enzymes-
substrate complex.
 Comparison of activation energies in the
uncatalyzed and catalyzed decompositions of
ozone.
Important Terms To Remember!
active site - a region of an enzyme comprised of
different amino acids where catalysis occurs or a small
portion of the surface of an enzyme which a specific
chemical reaction is catalyzed

substrate - the molecule being utilized and/or

modified by a particular enzyme at its active site

co-factor - organic or inorganic molecules that are

required by some enzymes for activity. These
include Mg2+, Fe2+, Zn2+ and larger molecules termed co-
enzymes like nicotinamide adenine dinucleotide (NAD+),
coenzyme A, and many vitamins.
Types of Enzymes

holoenzyme - a complete, catalytically active enzyme

including all co-factors OR an enzyme containing
a non protein group

apoenzyme - the protein portion of a holoenzyme

minus the co-factors OR the protein part of
holoenzyme

(holoenzyme = apoenzyme+cofactor)

isozyme - (or iso-enzyme) an enzyme that performs

the same or similar function of another enzyme
that occur in several different molecular forms.
Nomenclature of enzyme
Originally enzymes were given non descriptive names such as:
rennin : curding of milk to start cheese-making processor
pepsin : hydrolyzes proteins at acidic pH
trypsin : hydrolyzes proteins at mild alkaline pH

The nomenclature was later improved by adding the suffix -ase to the name of the substrate
with which the enzyme functions, or to the reaction that is catalyzed, for example:
Alcohol
dehydrogenase

Glucose isomerase

Glucose oxidase

Lactic acid
dehydrogenase
Enzyme reactions are different from
chemical reactions, as follows:
1. An enzyme catalyst is highly specific, and catalyzes only one or a
small number of chemical reactions. A great variety of enzymes exist,
which can catalyze a very wide range of reactions.

2. The rate of an enzyme-catalyzed reaction is usually much faster than

that of the same reaction when directed by nonbiological catalysts at mild
reaction condition.

3. A small amount of enzyme is required to produce a desired effect.

4. Enzymes are comparatively sensitive or unstable molecules

and require care in their use.
Enzymatic Reaction Principles
Biochemically, enzymes are highly specific for their
substrates and generally catalyze only one type of
reaction at rates thousands and millions times
higher than non-enzymatic reactions.

Two main principles to remember about enzymes are :

a) they act as CATALYSTS (they are not consumed in
a reaction and are regenerated to their starting state)

a) they INCREASE the rate of a reaction towards

equilibrium (ratio of substrate to product), but they
do not determine the overall equilibrium of a reaction.
 Reaction Rates
The rate of the reaction is effected by several factors
including the concentration of substrate,
temperature and pH.
For most standard physiological enzymatic reactions, pH
and temperature are in a defined environment (eg;
pH 6.9-7.4, 37oC).
This enzymatic rate relationship has been described
mathematically by combining the equilibrium constant,
the free energy change and first or second-order rate
theory.
Keq = eGo/RT
The net result for enzymatic reactions is that the lower
the activation energy, the faster the reaction rate,
and vice versa.
Specificity

Most synthetic catalyst are not specific i.e.,

they will catalyze similar reactions involving
many different kinds of reactants.

While enzymes are specific. They will

catalyze only one reaction involving only certain
substances.
Binding Energy
The interaction between enzyme and its substrate is
usually by weak forces.

In most cases, Van der Waals forces and hydrogen

bonding are responsible for the formation of ES
complexes.

The substrate binds to a specific site on the enzyme

known as the active site.
Classification of Enzyme
Enzymes fall into 6 classes based on function

1. Oxidoreductases: which are involved in oxidation, reduction, and

electron or proton transfer reactions

2. Transferases : transfer of functional group

3. Hydrolases : which cleave various covalent bonds by hydrolysis

4. Lyases : catalyse reactions forming or breaking double bonds

5. Isomerases : catalyse isomerisation reactions

6. Ligases : join substituents together covalently.

ENZYME KINETICS
Enzyme Kinetics
Mathematical models of single-substrate-enzyme-
catalyzed reactions were first developed by Henri in 1902
and Michaelis & Menten in 1913

Simple enzyme kinetics are now commonly referred to

as Michaelis-Menten or saturation kinetics

At high substrate concentrations, all active sites on the

enzyme are occupied by substrate enzyme is saturated

Models are based on data from batch reactors with

constant liquid volume in which the initial substrate, [S0],
and enzyme, [E0], concentrations are known
Enzyme kinetics deals with the rate of enzyme reaction

Kinetic studies of enzymatic reactions provide information

about :
(1)the basic mechanism of the enzyme reaction and
(2) other parameters that characterize the properties of the
enzyme.

The rate equations developed from the kinetic studies can

be applied in :
(1)calculating reaction time,
(2) yields, and
(3) optimum economic condition, which are important in the
design of an effective bioreactor.
Assume that a substrate (S) is converted to a product (P) with the help of an
enzyme (E) in a reactor as:

If you measure the concentrations of substrate and product with respect to

time, the product concentration will increase and reach a maximum
value, whereas the substrate concentration will decrease as shown in
Figure 2.1
The rate of reaction can be expressed in terms of either the change of the
substrate Cs or the product concentrations CP as follows:

change of the substrate Cs

product concentrations CP

Brown (1902) proposed that an enzyme forms a complex with its

substrate. The complex then breaks down to the products and
regenerates the free enzyme.

The mechanism of one substrate-enzyme reaction can be expressed

as:
One of the original theories to account for the
formation of the enzyme-substrate complex is the "lock
and key" theory.

The enzyme represents the lock and substrate

represents the key.

The main concept of this hypothesis is that there is a

topographical, structural compatibility between an
enzyme and a substrate which optimally favors the
recognition of the substrate as shown in Figure 2.3.
In multi substrate, enzyme-catalyzed reactions,
enzymes can hold substrates such that reactive
regions of substrates are close to each other
and to the enzymes active site, which is known as
the proximity effects (nearest in distance).

Multisubstrate
enzyme catalyst
reaction
Also, enzymes can hold substrates at certain positions
and angles to improve the reaction rate, which is
known as the orientation effect.

Alteration of active site

by activator
The reaction rate equation can be derived from
the preceding mechanism based on the following
assumptions:

1. The total enzyme concentration stays constant

during the reaction,

2. The amount of an enzyme is very small

compared to the amount of substrate. Therefore,
the formation of the enzyme substrate complex does not
significantly deplete the substrate.

3. The product concentration is so low that product

inhibition may be considered negligible.
Michaelis - Menten or saturation
kinetics
 Single-Substrate Enzyme Kinetics
It is assumed that:

1) The ES complex is
established very rapidly

2) The rate of the reverse

reaction of the second step is
negligible (i.e k-2~0)
Enzyme
Enzyme Product
Substrate Substrate
Complex (Assumption 2 is typically only
valid when product (P)
accumulation is negligible, at
the beginning of the reaction)
This model are based on data from batch reactors with
constant liquid volume in which the initial substrate,[S0],
and enzyme [E0], concentration are known.

An enzyme solution has a fixed number of active sites to

which substrate can bind. At high substrate
concentrations, all these sites may be occupied by
substrates or the enzyme is saturated.

Two major approaches used in developing a rate

expression for enzyme catalyzed reactions are ,
(1) rapid-equilibrium approach and
(2) quasi-steady-state approach.
 Rate of product formation:

Rate of variation of the ES complex:

Since the enzyme is not consumed,

the conservation equation yields,

At this point, an assumption is required in

order to achieve an analytical solution
 Assumption

(1)Rapid-Equilibrium
Assumption

(2) Quasi-Steady-State
Assumption
The Rapid Equilibrium Assumption
Henri and Michaelis and Menten used essentially this
approach.

Assuming equilibrium in the first part of the reaction (E+S

forms ES), we can use the equilibrium coefficient to express
[ES] in terms of [S]

The equilibrium constant is:

Since , if the enzyme is conserved, then

Substitution in

Michaelis-Menten
constant
Yields, (the prime () indicates
that it was derived
assuming rapid
equilibrium)
Where,
Low value:
enzyme has high
affinity for the
substrate
Vm= maximum forward rate of the
Corresponds to the
reaction (change with the addition of
substrate
additional enzyme but not addition of concentration, giving
substrate) half-maximal reaction
velocity.
Rate of Reaction as a Function of
Substrate Concentration
Quasi-Steady-State Assumption
Briggs and Haldane first proposed Quasi-steady-state
assumption

In a batch reactor at closed system, [E0] is considered

very small compared S

Therefore, d(ES)/dt 0

From equation d ES
k1 [ S ][ E ] k 1 k 2 ES ----1
dt

ES k1 [ E ][ S ]
----2
k1 k2
 Substituting , , and solve equation 2
for [ES],
ES k [ S ][ E0 ]
1 k 2
[S]
----3
k1

Production formation kinetics,

d[ S ] d[ P]
v
dt

dt
k 2 [ ES ] ----4

Substitute equation 3 into 4,

k 2 [ E0 ][ S ]
v ----5
k1 k2 / k1 [ S ]
Substituting,Vm k2 [ E0 ] and k 1 k 2
Km
k1
Vm [ S ]
v ----6
Km [ S ]

There is difference between Michaelis-Menten

constant [Km=k-1/k1] and Quasi-steady-
state constant [Km=k-1+k2/k1] .
Determination of Rate Parameters for
Michaelis-Menten Type Kinetics

Lineweaver-Burk plot
Eadie- Hofstee plot
Hanes-Woolf plot
Batch kinetics
1. Lineweaver-Burk Plot

From equation 6 (Quasi-steady-state ),

Vm [ S ]
v
Km [ S ]
Double reciprocal plot
slope

Y-intercept

Lineweaver-Burk plot gives good estimates on Vm but not

necessarily on Km (error relates with substrate conc)
2. EadieHofstee Plot
Vm [ S ]
From equation 6, v
Km [ S ]

Rearranged equation 6,

plot v versus v/[S] gives a line

of slope Km and y-axis
intercept of Vm

Can be subject to large errors since both coordinates

contain v, but less bias on points at low [S]
3. HanesWoolf plot
Rearrangement of equation 6 yields,

slope
intercept

This plot is used to

determine Vm more
accurately.
4. Batch kinetics
Integration of equation 6 and rearranged yields,
1 [ So ] 1 [ So ] [ S ] Vm
ln
t [S] Km t Km
slope
Y-intercept

1 [ So ]
ln
t [S]

[ So ] [ S ]

t
Lets Understand it More
based on your first
Experiment ERT 317
Experiment 1
Effect of Substrate Concentration on
Enzyme Kinetics Study

OBJECTIVES
1.To develop a suitable standard curve for enzyme
assay
2.To analyze the effect of substrate concentration on
the activity of enzyme
3.To determine Vmax and Km from the enzyme reaction
using enzyme kinetics plots.
 COURSE OUTCOMES
CO1 Ability to develop enzyme reactions based
on its kinetics study and applied catalysis

INTRODUCTION
The enzyme -amylase can catalyze the hydrolysis of internal -1,4-glycosidic bond
present in starch with the production of reducing sugars. In the study of substrate
concentration on enzyme kinetics, the enzyme is kept constant where as the
concentration of starch is taken in increasing order. As the substrate concentration
increases, the amount of products produced in every successive tube also increases.
This enzyme-substrate reaction can be determined by measuring the increase in
reducing sugars using the 3,5-dinitrosalicylic acid reagent. In an alkaline condition, the
pale yellow coloured the 3,5-dinitrosalicylic acid undergo reduction to yield orange
coloured 3-amino-5-nitrosalicylic acid. The absorbance of resultant solutions is read at
540nm. The intensity of colour depends on the concentration of reducing sugars
produced.
 hydrolysis
Starch + -amylase Maltose + glucose

Amylose comprises 15-30% of the common starches.

Amylose is a linear polymer containing up to 6000
glucose units, connected by (1, 4) linkages.

Therefore, we use -amylase to hydrolyzed starch

in this hydrolysis process. The hydrolysis of starch
with a low molecular weight, catalyzed by an - amylase,
is one of the most important commercial enzyme
processes.
 hydrolysis
Starch + -amylase Maltose + glucose

What does alpha amylase do?

-Amylase is a protein enzyme EC 3.2.1.1 that

hydrolyses alpha bonds of large, alpha-linked
polysaccharides, such as starch and glycogen,
yielding glucose and maltose.

The individual subunits that make

up maltose are glucose
 hydrolysis
Starch + -amylase Maltose + glucose

Your Task: 1) Develop of maltose standard curve

why? How?
-to measure the product of reducing sugar (after
hydrolysis process), we NEED a standard.
Pure Sugar : Maltose (main reducing sugar produced
from starch after enzymatic hydrolysis)
Reagent needed: DNS reagent (the intensity of colour
in DNS depends on the concentration of reducing
sugars produced)
 Simple Method:
1. Prepare at least 5 different concentration of pure
sugar (Maltose)
2. Prepare 1 Blank (no sugar/no maltose)
3. All samples + DNS reagent (follow DNS method)
4. Read absorbance @ 540 nm (for blank: Zeroing)
5. Plot Absorbance reading (y-axis) VS Maltose conc
(x-axis)
6. Linear graph with an equation Y=mx+c (make sure
R2 for any standard curve is 0.999)
Your Task: 2) Study effect of substrate concentration
on enzyme activity

-Vary substrate (starch) concentration but fix the enzyme concentration.

SIMPLE METHOD:
1. Run the hydrolysis process (different starch conc react with an enzyme)
at 37oC, 3 min. Produced product (hydrolysate) containing reducing
sugars (maltose)
Blank: no starch
1. All product samples contain unknown conc of maltose. Therefore, we
need to run DNS method.
2. Samples + DNS reagent (follow DNS method) .
3. Cek concentration of samples using MALTOSE Standard Curved.
 Starch concentration, Abs at 540 nm Amount of Velocity [V] in 1/[V] 1/[S]
[S] (%) maltose moles/min
(g)
0.02
0.04
0.06
0.08
0.10

1.Draw the Michaelis-Mentens plot and Line Weaver Burk plot

using the data in Table 1.3 and find the Vmax and Km from the plot.

1.Compare the value of Vmax and Km from both plot and give your
reason of their differences.
Michaelis-Mentens plot
Lineweaver-Burk Plot

From equation 6 (Quasi-steady-state ),

Vm [ S ]
v
Km [ S ]
Double reciprocal plot
slope

Y-intercept

Lineweaver-Burk plot gives good estimates on Vm but not

necessarily on Km (error relates with substrate conc)
Thank You