MODULE 2:

ENZYMOLOGY
Enzymes have been significant industrial products for over a century. The range of
potential application is still increasing rapidly. With the advent of recombinant DNA
technology, it has become possible to make formerly rare enzymes in large quantities and,
hence, reduce cost.

Technological advances have facilitated the use of enzymes over an increasingly

broad range of process conditions. Enzymes from organisms that grow in unusual
environments (e.g. deep ocean, salt lakes, hot springs, and industrial waste sites) are
increasingly available for study and potential use. New enzymes and better control of
reaction conditions allow the use of enzymes in the presence of high concentrations of
organics, in high salt aqueous environments, or at extreme temperatures, pH, or pressures.
As we couple new insights into the relationship of enzyme structure to biological function
with recombinant DNA technology, we are able to produce enzymes that are human
designed or manipulated (in reference to Module 1). We no longer need to depend solely
on natural sources for enzymes.

Since enzymes are important in various chemical process industries it is only

appropriate that as future chemical engineers, you will be equipped with the basic
information and concept on enzymes.

At the end of this module, you should be able to apply enzyme kinetics in commercial
techniques and formulate and solve problems on stoichiometry, kinetics and design of
bioreactors.

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 Unit 1: Introduction to Enzymology
Engage

Enzymology is a branch of biochemistry that deals with the properties,

activity and significance of enzymes.

Studies on enzymes have been developed and are still developing throughout the
years. Listed below are some of the most important breakthroughs in enzymology.
 1897: Buchner first extracted active enzymes from living cells.
The experiment for which Buchner won the Nobel Prize consisted of producing a cell-
free extract of yeast cells and showing that this “press juice” could ferment sugar. The
cell-free extract was produced by combining dry yeast cells, quartz and kieselguhr
and then pulverizing the yeast cells with a pestle and mortar. This mixture would then
become moist as the yeast cells’ contents would come out of the cells. Once this step
was done, the moist mixture would be out through a press and the resulting “press
juice” had glucose, fructose, or maltose added and carbon dioxide was seen to
evolve. Microscopic investigation revealed no living yeast cells in the extract. Buchner
hypothesized that yeast cells secrete proteins into their environment in order to
ferment sugars, but it was later found that fermentation occurs inside the yeast cells.
 1926: The first successful isolation of a pure enzyme was achieved by Sumner. The
enzyme he worked with was urease, which he isolated from jack beans. He
accomplished this by mixing purified urease with acetone and then chilling it; the
chilled solution produced crystallized urease. He was also able to show by chemical
tests that his pure urease was a protein. This was the first experimental proof that an
enzyme is a protein, a controversial question at the time.

Molecular Structure of Urease

Digital Image. Wikipedia, 08 May 2020, https://en.wikipedia.org/wiki/Urease

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 Explore

Enzymes are biological catalysts that are protein molecules in nature

and are produced by living cells (animal, plant and microorganism).

Enzymes are absolutely essential as catalysts in biochemical reactions. They have high
molecular weights, 15,000 to several million daltons (1 dalton = 1 g/mol).

Almost every reaction in a cell requires the presence of a specific enzyme. 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.

Examples of biological processes in the body with the aid of enzymes.

1. Digestion
In order to break down large molecules of food to smaller molecules faster, enzymes
called hydrolases are used. These hydrolases are pepsin and trypsin.
2. DNA Copying
Your cells contain genetic material called chromosomes, each carrying an array of
genes that encode your individual genetic makeup. This genetic material is in the
form of a molecule called deoxyribonucleic acid, or DNA. When cells in your body
divide, each newly created cell must contain an exact copy of your DNA. To carry
out this copying, or replication, your body relies on specific enzymes. For example,
enzymes called helicases and gyrases unwind the tightly coiled DNA strands so they
can be copied. DNA polymerase enzymes participate in the actual copying process.
DNA ligase enzymes are involved in finalizing the copying process.
3. Energy Production
Your body must generate energy to carry out all of its functions. Although blood sugar,
or glucose, is the preferred fuel to generate energy, proteins and fats can also be
used. These complex processes -- collectively known as cellular respiration -- rely on
enzymes and generate molecules called adenosine triphosphate (ATP) that power
your cells. For example, energy generation using glucose is called glycolysis. Each of
the 10 steps in glycolysis involves a different enzyme. Each molecule of glucose
broken down by glycolysis yields 2 ATP molecules. Examples of enzymes used in
glycolysis include hexokinase, aldolase, enolase and pyruvate kinase. Other enzymes
are involved in the generation of ATP from proteins and fats.

A catalyst increases the rate of a chemical reaction without undergoing a permanent

chemical change. Also, a catalyst influences the rate of a chemical reaction but does not
affect reaction equilibrium. Equilibrium can be calculated using only the thermodynamic

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 properties of the substrates (substrate is the biochemist‘s term for what we usually call a
reactant) and products. Reaction kinetics, however, involve molecular dynamics and are
presently impossible to predict accurately without experimental data.

The catalytic ability of enzymes is due to its particular protein structure. A specific
chemical reaction is catalyzed at a small portion of the surface of an enzyme, which is known
as the active site. Some physical and chemical interactions occur at this site to catalyze a
certain chemical reaction for a certain enzyme. The substrate (a biochemist’s term for
reactant) binds to the active site.

Image Representation of an Enzyme

Digital Image. DNA of Bioscience, 19 May 2016, http://dnaofbioscience.blogspot.com/2016/05/what-is-active-site-of-

enzyme.html

The figure below shows the structure and active sites of one alchoholdehydrogenase.
While enzymes are specific in function, the degree of specificity varies. Some may act on
closely related substrates and are said to exhibit group specificity; other are more exacting

Shijie Liu, (2013), Bioprocess Engineering, Amsterdam: Elsevier B.V., P. 326

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 in their substrate requirements and are said to be absolutely specific. The product formed
from a particular enzyme and substrate is also unique. Enzymes are able to distinguish
between stereochemical forms and only one isomer of a particular substrate may be
catalyzed to react.

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 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.
3. The reaction conditions (temperature, pressure, pH, and so on) for the enzyme
reactions are very mild.
4. Enzymes are comparatively sensitive or unstable molecules and require care in their
use.

For design and analysis of a reacting system, we must have a mathematical formula
which gives the reaction rate (moles reacted per unit time per unit volume) in terms of
composition, temperature, and pressure of the reaction mixture.
If you have studied catalyzed reactions before, you have seen a strategy for obtaining
a reasonable reaction rate expression. First an educated guess is made about what
elementary reactions go on at the molecular scale. Then, assuming that one of these
molecular events is slow relative to all the rest, an expression for obtaining a reasonable
reaction rate expression is made. We shall follow precisely this approach in this chapter’s
quest for rate expressions for enzyme-catalyzed reactions.

Nomenclature of Enzymes
Originally enzymes were given non-descriptive names such as:
rennin : curding of milk to start cheese-making process
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:
Name of enzyme = Name of substrate + -ase

1. Chemical reaction: starch ⟶ glucose + maltose + oligosaccharides

Substrate: Starch (Amylum)
Name of enzyme: α – amylase

2. Chemical reaction: lactose ⟶ glucose + galactose

Substrate: lactose
Name of enzyme: lactase

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 3. Chemical reaction: fat ⟶ fatty acids + glycerol
Substrate: fat (lipid)
Name of enzyme: lipase

4. Chemical reaction: maltose ⟶ glucose

Substrate: maltose
Name of enzyme: maltase

5. Chemical reaction: Urea + H2O ⟶ 2 NH3+ CO2

Substrate: urea
Name of enzyme: urease

6. Chemical reaction: cellobiose ⟶ glucose

Substrate: cellobiose
Name of enzyme: cellobiase

For example:
Name of enzyme = reaction which is catalyzed + -ase

1. Chemical reaction: ethanol + NAD+ ⇌ acetaldehyde + NADH2

Name of reaction: alcohol dehydrogenation
Name of enzyme: alcohol dehydrogenase

2. Chemical reaction: glucose ⇌ fructose

Name of reaction: glucose isomerization
Name of enzyme: glucose isomerase

3. Chemical reaction: D − glucose + O2 + H2O ⟶ gluconic acid

Name of reaction: glucose oxidation
Name of enzyme: glucose oxidase

4. Chemical reaction: lactic acid ⟶ pyruvic acid

Name of reaction: lactic acid dehydrogenation
Name of enzyme: lactic acid dehydrogenase

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 As more enzymes were discovered, this system generated confusion and resulted in
the formation of a new systematic scheme by the International Enzyme Commission in 1964.
The new system categorizes all enzymes into six major classes depending on the general
type of chemical reaction which they catalyze. Each main class can be designated by a
numerical code system.

The table below shows the six main classes of enzymes, 1st digit, and the rest of the columns
are the 2nd, 3rd and 4th digit.

1st digit (class) 2nd digit 3rd digit 4th

1. Oxidoreductases Donor of hydrogen or electron Acceptor of hydrogen
Transfer of hydrogen 1 Alcohol or electron
or oxygen atoms or 2 Aldehyde or ketone 1 NAD+ or NADP+ or
electrons from one 3 Alkene, -CH=CH- peroxide
substrate to another: 4 Primary Amine: CH-NH2 2 cytochrome or heme
oxidases or 5 Secondary Amine: CH-NH protein
dehydrogenases 6 NADH or NADPH 3 O2
7 Nitrogenous compound 4 S-S
8 sulfur group 5 quinone or similar
9 heme group 6 nitrogenous group
10 diphenols or related 7 iron-sulfur-protein
11 peroxide 8 flavin
12 hydrogen 11 incorporating two O
13 single donor with O2 12 incorporating one O
14 paired donors, with O2 98 other, known
15 acting on superoxide 99 other
16 oxidizing metal ion
17 acting on CH or CH2 groups
18 iron-sulfur-protein
19 reduced flavodoxin
20 phosphorous or arsenic
21 on XH and YH to form XY
97 oxidoreductases
98 other, using H2 as reductant
99 other, using O2 as oxidant
2. Transferases General type of group Nature of group
Group transfer 1 1-Carbon group transferred
excluding hydrogen or 2 aldehyde or ketone
oxygen: 3 acyl group –COR

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 AX + B → BX + A 4 glycosyl group
5 Alkyl or aryl, other than methyl
6 nitrogenous groups
7 phosphate group
8 sulfur-containing grouop
9 selenium-containing groups
3. Hydrolases Type of bond hydrolyzed
Catalyzes hydrolysis: 1 ester
A-X + H2O → X-OH + 2 glycosylases
HA 3 ether
4 peptide
5 C-N bonds other than
peptides
6 acid anhydrides
7 C-C
8 halide
9 phosphorus-nitrogen
10 S-N
11 C-phosphorus
12 S-S
13 S-S
4. Lyases Type of bond broken Group removed
Nonhydrolytic removal 1 C-C 1 Carboxyl
of groups with product 2 C-O 2 Aldehyde
usually contains 3 C-N 3 Ketoacid
double bond 4 C-S
5 C-Halide
6 P-O
99 Other
5. Isomerases Type of reaction Type of molecule
1 Racemizationor epimerization 1 amino acids
2 cis-trans isomerizations 2 hydroxyacids
3 intramolecular 3 carbohydrates
oxidoreductases
4 intramolecular transfer
reactions
5 intramolecular lyases
99 other
6. Ligases Type of bond formed
Synthesis of bonds with 1 C-O
breaking down of ATP 2 C-S
or nucleoside 3 C-N
triphosphates 4 C-C
5 phosphate ester
6 N-metal

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 For example:
Trivial Name: Alcoholdehydrogenase
Systematic Name: Alcohol. NAD+.oxidoreductase.
Enzyme Code: EC 1.1.1.1

Cofactors

Another distinguishing characteristic of enzymes is their frequent need for cofactors.

A cofactor is a nonprotein compound which combines with an otherwise inactive protein
(the apoenzyme) to give a catalytically active complex. While this complex is often called
a holoenzyme by biochemists, we shall often simply call it an enzyme. Two distinct varieties
of cofactors exist. The simplest cofactors are metal ions. On the other hand, a complex
organic molecule called a coenzyme may serve as a cofactor. Often these cofactors are
bound relatively loosely to the enzyme, and there is equilibrium between enzyme,
apoenzyme, and cofactor.

Enzyme (Holoenzyme) = Apoenzyme + Cofactor

Example of Some Cofactors with paired Enzyme

Cofactor Enzyme
Zn2+ Alcohol dehydrogenase
Carbonic anhydrase
Carboxypeptidase
Mg 2+ Phosphohydrolases
Phosphotransferases
Mn2+ Arginase
Phosphotransferases
Fe2+ or Fe3+ Cytochromes
Peroxidase
Catalase
Ferredoxin
Cu (Cu ) Tyrosinase
2+ +

Cytochrome oxidase
K+ Pyruvate phosphokinase (also requires
Mg2+)
Na Plasma membrane ATPase (also requires
+

K+ and Mg2+)

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 Catalysts Activity

Both synthetic and biological catalysts can gradually lose activity as they participate
in chemical reactions. As the table in the below illustrates, some of these deactivation
processes are conceptually and physically similar. Still, enzymes are in general far more
fragile. While their complicated, contorted shapes in space often endow enzymes with
unusual specificity and activity, it is relatively easy to disturb the native conformation and
destroy the enzyme’s catalytic properties.

Some related Deactivation Phenomena in Catalysis by Enzymes or Solid Surfaces

Phenomena Cause
Solid Catalysts
Product Inhibition (reversible) Accumulating product occupies active
sites, decreasing overall reaction rate.
Catalyst poisoning (irreversible) The active site reacts essentially
irreversibly with some species to yield an
inactive catalyst site.
Catalyst sintering (irreversible) A high surface area catalyst loses active
surface area resulting in a decrease in
the number of active site per catalyst
volume.
Enzyme Catalysts
Product inhibition Same as above
Catalyst poisoning Same as above
Catalyst denaturation The enzyme molecule conformation
changes sufficiently to result in a loss of
the catalyst activity

Turnover Number

It is often asserted that enzymes are more active, i.e., allow reactions to go faster,
than nonbiological catalysts. A common measure of activity is the turnover number, which
is the net number of substrate molecules reacted per catalyst site per unit time. The table in
the next page gives the turnover numbers for several reactions, which shows that at the
ambient temperatures where enzymes are most active they are able to catalyze reactions
faster than the majority of artificial catalysts. When the reaction temperature is increased,
however, solid catalysts may become as active as enzymes. Unfortunately, enzyme activity
does not increase continuously as the temperature is raised. Instead, the enzyme typically
denatures at quite a low temperature, often only slightly above that at which it is typically
found.

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 Some turnover numbers for enzyme and solid catalyzed reactions
Enzyme Range of reported values
at 0-37°C
Ribonuclease 2 – 2x103
Trypsin 3x10-3 – 1x102
Papain 8x10-2 – 1x101
Bromelain 4x10-3 – 5x10-1
Carbonic anhydrase 8x10-1 – 6x105
Fumarate hydratase 1x103 (forward)
3x103 (reverse)

Some turnover numbers for some solid-catalyzed reactions

Solid Catalyst N Temp., °C
SlO2 – Al2O3 (impregnated) 3x10-8 25
2x10 4 420
Decationized zeolite ~103 25
~108 325
V2O3 7x10-11 25
102 350
Treated Cu3Au 2x107 25
3x10 10 327
AlCl3 – Al2O3 1x10-2 25
1.x10 -2 60

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 Explain

Enzymes are protein, glycoprotein, or RNA molecules that catalyze biologically

important reactions. Enzymes are very effective, specific, and versatile biocatalysts.
Enzymes bind substrate molecules and reduce the activation energy of the reaction
catalyzed, resulting in significant increases in reaction rate.

Further Reading:

Chapter 8, Section 8.1, Bioprocess Engineering by Shijie

Liu (soft copy of the book is present in our google
classroom)

Elaborate

Commercial Applications of Enzyme

Enzymes have been used since early human history without knowledge of what they were
or how they worked. They were used for such things as:
• Making sweets from starch
• Clotting milk to make cheese
• Brewing soy sauce
Enzymes have been utilized commercially since the 1890s, when fungal cell extracts were
first added to brewing vats to facilitate the breakdown of starch into sugars (Eveleigh, 1981).
The fungal amylase takadiastase was employed as digestive aid in the United States as early
as 1894.

Further Reading:
Chapter 2, Section 2.1.2, Fundamentals of Biochemical
Engineering by James M. Lee (soft copy of the book is
present in our google classroom)

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 Evaluate

Enzymes are important elements in industrial processes. They determine the success of the
process and the quality of products produced.

Evaluative Assessment:

Discussion forum via google classroom.

References

James M. Lee (2008). Fundamentals of Biochemical Engineering. Springer.

Shijie Liu (2013). Bioprocess Engineering. Elsevier B.V.

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 MODULE 2:
ENZYMOLOGY

Unit 2: Enzyme Kinetics

Engage

A mathematical model of the kinetics of single substrate-enzyme-catalyzed reactions

was first developed by V. C. R. Henri in 1902 and by L. Michaelis and M. L Menten in 1913.
These models are based on data from batch reactors with constant liquid volume in which
the initial substrate, [S]0, and enzyme, [E]0, concentrations are known. More complicated ES
interactions such as multisubstrate - multienzyme reactions can take place in biological
systems.

Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by
various chemical and physical conditions. Kinetic studies of enzymatic reactions provide
information about the basic mechanism of the enzyme reaction and other parameters that
characterize the properties of the enzyme. The rate equations developed from the kinetic
studies can be applied in calculating reaction time, yields, and optimum economic
condition, which are important in the design of an effective bioreactor.

Review:

Basic terms and concepts in Chemical Reaction

Engineering (Kinetics) on reaction rates.

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 Explore

Simple Enzyme Kinetics

Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E) in
a reactor as:
𝐄
𝐒→𝐏

The rate of reaction can be expressed in terms of either the change of the substrate
concentration Cs or the product concentrations Cp as follows:
𝐝𝐂𝐬
𝐫𝐒 = −
𝐝𝐭
𝐝𝐂𝐏
𝐫𝐏 =
𝐝𝐭

The prior equations given are the slopes of the curves as shown below.

The change of product and substrate concentrations with respect to time

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 In order to understand the effectiveness and characteristics of an enzyme reaction,
it is important to know how the reaction rate is influenced by reaction conditions such as
substrate, product, and enzyme concentrations. If we measure the initial reaction rate at
different levels of substrate and enzyme concentrations, we obtain a series of curves like the
one shown on the below.

It has been shown experimentally that if the amount of the enzyme is kept constant
and the substrate concentration is then gradually increased, the reaction velocity will
increase until it reaches a maximum. After this point, increases in substrate concentration will
not increase the velocity. The reaction rate remains constant.

The kinetic parameters Km and rmax are important in order to have a quantitative
value for the performance of an enzyme. Km (Michaelis-Menten constant) is the substrate
concentration at half of rmax. While, rmax is the maximum reaction rate wherein even if you
increase the substrate concentration it will no longer be affected.

From the curve, the following can be concluded:

1. The reaction rate is proportional to the substrate concentration (that is, first-order
reaction) when the substrate concentration is in the low range.
2. The reaction rate does not depend on the substrate concentration when the
substrate concentration is high since the reaction rate changes gradually from first
order to zero order as the substrate concentration is increased.
3. The maximum reaction rate rmax is proportional to the enzyme concentration within
the range of the enzyme tested.

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 Henri observed this behavior in 1902 (Bailey and Ollis, p. 100, 1986) and proposed the rate
equation.

The Michaelis-Menten Equation:

𝑟𝑚𝑎𝑥 𝐶𝑠
𝑟𝑃 =
𝐾𝑚 + 𝐶𝑠

Where:
rp = reaction rate
Cs = substrate concentration
rmax and KM are kinetic parameters which have to be determined experimentally.

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
𝒌𝟏
𝑺 + 𝑬 ↔ 𝑬𝑺
𝒌𝟐
𝒌𝟑
𝑬𝑺 → 𝑷 + 𝑬

Brown’s kinetic inference of the existence of the enzyme-substrate complex was

made long before the chemical nature of enzymes was known, 40 years before the
spectrophotometric direction of such complexes.

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

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 Schematic of lock-and-key theory
Digital Image. Difference Between, 24 January 2018, https://www.differencebetween.com/difference-between-induced-fit-
and-lock-and-key/

Demonstration of lock-and-key theory

Digital Image. Wikibooks, 08 September 2019, https://en.wikibooks.org/wiki/Principles_of_Biochemistry/Enzymes

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, that is, 𝐶𝐸0 = 𝐶𝐸𝑆 +
CE .
Where:
CE0 = initial enzyme concentration
CES = enzyme concentration at ES complex
CE = free enzyme concentration

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 2. The amount of enzyme is very small compared to the amount of substrate. Therefore,
the formation of enzyme-substrate complex does not significantly deplete the
substrate.
3. The product concentration is so low that product inhibition may be considered
negligible.

In addition to the preceding assumptions, there are three different approaches to derive
the rate equation:
1. Michaelis-Menten approach / Fast Equilibrium Step Assumption (Henri and Michaelis
and Menten):
It is assumed that the product-releasing step is much slower than the reversible
reaction and the slow step determines the rate, while the other is at equilibrium. This is an
assumption which is often employed in heterogeneous catalytic reactions in chemical
kinetics. Even though the enzyme is soluble in water, the enzyme molecules have large and
complicated three-dimensional structures. Therefore, enzymes can be analogous to solid
catalysts in chemical reactions. Furthermore, the first step for an enzyme reaction also
involves the formation of an enzyme-substrate complex, which is based on a very weak
interaction. Therefore, it is reasonable to assume that the enzyme-substrate complex
formation step is much faster than the product releasing step which involves chemical
changes.

2. Briggs-Haldane approach / Pseudosteady-State Hypothesis (G.E. Briggs and J.B.S.

Haldane):
The change of the intermediate concentration with respect to time is assume to be
negligible, that is 𝑑𝐶ES/ 𝑑𝑡 = 0. This is also known as the pseudo-steady-state (or quasi-steady-
state) assumption in chemical kinetics and is often used in developing rate expressions in
homogeneous catalytic reactions.

3. Numerical solution: Solution of the simultaneous differential equations developed

without simplification.

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 Explain
How Enzymes Work

Enzymes lower the activation energy of the reaction catalyzed by binding the
substrate and forming an enzyme-substrate complex. Enzymes do not affect the free-energy
change or the equilibrium constant.

The molecular species of enzyme-substrate (ES) interaction differ for different enzyme
and substrate pairs. Various studies using crystallography, X-ray, and Raman spectroscopy
have revealed the presence of the ES complex. The interaction between the enzyme and
its substrate is usually by weak forces. When the substrate enters the active site of an enzyme
it will be held initially by noncovalent forces. These noncovalent forces responsible fo binding
may be employed to lower the activation energy of the reaction. The type of noncovalent
forces that are involved can be summarized as follows:

a. Electrostatic interactions
These include charge-charge (inversely proportional to the distance between the
charged enzyme active site to the charged substrate), dipole-dipole, charged-
induced dipole, and dipole-induced dipole interactions. The magnitude of these
forces depends on the distance between molecules, all depend inversely on the
dielectric constant of the solvent between the ions or dipoles.

b. Van der Waals forces

Comprised of electron cloud repulsion and attractive dispersion forces (London
forces). Dispersion forces are not large, but in an enzyme, the sum of all such forces
between substrate and enzyme may be quite significant.

c. Hydrogen bonds
Important in biological systems and occur when two electronegative atoms are ound
to a common proton. Often oxygen is one of the atoms.

d. Hydrophobic forces
These reflect the tendency of apolar molecules to partition from an aqueous
environment to a hydrophpbic one. The driving force for such movement can be
thought of as a result of the entropy gain when water molecules, which must be
structured around an apolar molecule, are able to assume a more random
arrangement when the molecule is transferred. The magnitude of this force is found
experimentally to depend on the surface area of the molecule.

When the substrate moves from the external aqueous environment to the active site
of the enzyme, its salvation shell is lost and one or more of the above forces are important in

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 determining the strength of its binding. Hydrogen bonding and electrostatic interactions are
generally most important. The difference in energies between the solvated state and that of
the ES complex determines the strength of substrate binding. The substrate binds to the
active site. The substrate is a relatively small molecule and fits into a certain region on the
enzyme molecule, which is a much larger molecule, as modelled by the lock-and-key
theory.

In multisubstrate enzyme-catalyzed reactions, enzymes can hold substrates such that

reactive regions of substrates are close to each other and to the enzyme’s active site, which
is known as the proximity effect. This is the simplest mechanism by which an enzyme may
enhance the rate of a reaction. The reactants were brought at the active sites and would
then be present at local concentrations that are much higher than those present in solution.
How large a rate enhancement might we expect from such approximation? Some insight
into this can be obtained by examining intramolecular catalysis. Here one group adjacent
to a reacting group provides catalytic assistance in the reaction.

An example of intramolecular catalysis is provided by the hydrolysis of tetramethyl

succinanilic acid. This reaction is illustrated as

The carboxylic acid moiety provides intramolecular assistance in the hydrolysis of the amide
bond. At pH 5, the reaction occurs with a half-life of 30 min, whereas the corresponding
hydrolysis of the unsubstituted acetanilide is some 300 years! This difference in rates
corresponds to a rate enhancement of 1.6x108. Unsubstituted succinanilic acid is hydrolyzed
1200 times more slowly than the tetramethyl substituted compound; the methyl groups are
important in bringing the catalytic group close to the reacting amide bond.

In enzyme-catalyzed reactions, this enhanced local concentration effect can

account for some of the rate enhancement but is generally not sufficiently large. It does
provide a lower limit to the rate acceleration that might be expected however. We must
turn to other mechanisms to provide an explanation for the catalytic abilities of enzymes.

Covalent Catalysis: Electrophilic and Nucleophilic Catalysis

An enzyme can form a covalent bond with one or more reactants and so alter the
reaction path from that observed in the uncatalyzed case. The discovery that enzymes may
indeed form covalent intermediates relied on early kinetic observations, including “burst”
kinetics and the observation of constant rates of product release from substrates with varying

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 substituents. Today, the crystallographic structures of many enzymes and their substrate-
containing intermediates are well-known, providing further evidence for the formation of
such covalent intermediate compounds.

Acids and Bases

Acids and bases can catalyze reactions by either donating or accepting a proton
which is transferred in the transition state. When such a charged group develops in the
transition state, the resulting positive or negative charge makes the transition state
unfavorable. The presence of acids or bases results in a stabilization of such a transition state.
By providing acid or base groups at the active site, an enzyme is thus able to stabilize the
charged transition state. This mechanism is employed by a wide variety of enzymes.

Electrostatic Catalysis

In water, the large dielectric constant results in a small electrostatic interaction energy
between charges, and electrostatic catalysis is not generally important in homogeneous
catalysis in aqueous systems. However, the active site of a protein is very heterogeneous,
and the dielectric constant of the medium between charged groups may be quite different
from water. The aromatic and aliphatic amino acid residues present at the active site act to
reduce the dielectric constant and charged amino acid residues act as fixed dipoles, thus
stabilizing charge quite effectively. The electrostatic interaction energy depends on the
charges and is inversely proportional to the dielectric constant and distance between
charges.

Catalysis Involving Metal Ions

In metalloenzymes, a metal ion is present at the active site, and this ion plays an
important role in stabilizing negative charges that are formed in electrophilic catalysis. Zinc,
copper, and cobalt are commonly involved in coordination of oxyanions involved as
reaction intermediates.

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 Elaborate

Sample Problem

When glucose is converted to fructose by glucose isomerase, the slow product

formation step is also reversible as:
𝑘1
𝑆 + 𝐸 ↔ 𝐸𝑆
𝑘2
𝑘3
𝐸𝑆 ↔ 𝑃 + 𝐸
𝑘4
Derive the rate equation by employing (a) the Michaelis-Menten and (b) the Briggs-
Haldane approach. Explain when the rate equation derived by the Briggs-Haldane
approach can be simplified to that derived by the Michaelis-Menten approach.

Evaluate

Problem Set:

Solve the following problems and show complete solution. Box all final
answers:
1. Suppose the following sequence describes the reactions of two
different substrates catalysed by one enzyme: Derive the rate equation
by making the Michaelis-Menten assumption.
𝑘1
𝐸 + 𝑆1 ↔ 𝐸𝑆1
𝑘2
𝑘3
𝐸𝑆1 + 𝑆2 ↔ 𝐸𝑆1 𝑆2
𝑘4
𝑘5
𝐸𝑆1 𝑆2 → 𝐸 + 𝑃

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 2. Chymotrypsin is a serine protease that cleaves the amide linkages in
proteins and peptides. It has a binding pocket which is selective for the
aromatic residues of amino acids. The reaction occurs by the reversible
formation of a Michaelis-Menten complex, followed by acylation of Ser-
195 to give a tetrahedral acyl enzyme intermediate. Chymotrypsin will
also act as an esterase. The reaction steps simplified as
𝑘1
𝐸 + 𝑆1 ↔ 𝐸𝑆1
𝑘2
𝑘2
𝐸𝑆1 → 𝐸𝑆2
𝑘3
𝐸𝑆2 → 𝐸 + 𝑃
Derive the rate of formation of P that is consistent with this mechanism.

References

James M. Lee (2008). Fundamentals of Biochemical Engineering. Springer.

Shijie Liu (2013). Bioprocess Engineering. Elsevier B.V.

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 MODULE 2:
ENZYMOLOGY

Unit 3: Determination of Kinetic Parameters

Engage

To fully specify the kinetics of Michaelis-Menten reactions, two rate constants, rmax
and Km, must be evaluated.

Km (Michaelis-Menten constant) is the substrate concentration at half

of rmax. While, rmax is the maximum reaction rate wherein even if you
increase the substrate concentration it will no longer be affected.

Estimating kinetic parameters for M-M reactions is not as straightforward as for zero-
and first-order reactions. Several graphical methods are available; unfortunately some do
not give accurate results.

Explore

The first step in kinetic analysis of enzyme reactions is to obtain data for rate of
reaction, r, as a function of substrate concentration, Cs. Rate of reaction can be determined
from batch concentration data. Typically, only initial rate data are used. This means that
several batch experiments are carried out with different initial substrate concentrations; from
each set of data the reaction rate is evaluated at time zero. The results are plotted
graphically so that the validity of the kinetic model can be tested and the values of the
kinetic parameters can be estimated.
The most straightforward way is to plot r against Cs.

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 The asymptote for r will be rmax and KM is equal to CS when r = 0.5rmax. However, this
is an unsatisfactory plot in estimating rmax and KM because it is difficult to test the validity of
the kinetic model. Therefore, the Michaelis-Menten equation is usually rearranged so that
the results can be plotted as a straight line.

The Michaelis-Menten equation is rearranged to be expressed in linear form. Some of the

better known methods are:
a. Langmuir plot
b. Lineweaver-Burk plot
c. Eadie-Hofstee plot

Langmuir Plot
 Also known as Hanes-Woolf plot
 Proponents of this method are
Charles Samuel Hanes and Barnet
Woolf
 This equation was given by
Langmuir for the treatment of
data from the adsorption of gas
on a solid surface.
 The linearized form of the M-M
equation is:

𝑪𝒔 𝑲𝒎 𝟏
= + 𝑪𝒔
𝒓 𝒓𝒎𝒂𝒙 𝒓𝒎𝒂𝒙

Lineweaver-Burk Plot

 Also known as double

reciprocal plot
 Proponents are Hans
Lineweaver and Dean Burk
 The linearized form of the M-M
equation is:

𝟏 𝟏 𝑲𝒎 𝟏
= +
𝒓 𝒓𝒎𝒂𝒙 𝒓𝒎𝒂𝒙 𝑪𝒔

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 Eadie-Hofstee Diagram
 Also known as Woolf-Eadie-Augustinson-
Hofstee or Eadie-Augustinson
 The linearized form of the M-M equation
is:
𝒓
𝒓 = 𝒓𝒎𝒂𝒙 − 𝑲𝒎
𝑪𝒔

The Lineweaver-Burk plot is more often employed than the other two plots because it
shows the relationship between the independent variable CS and the dependent variable r.
However, 1/r approaches infinity as CS decreases, which gives undue weight to inaccurate
measurements made at low substrate concentrations, and insufficient weight to the more
accurate measurements at high substrate concentrations. The points on the line in the figure
for Lineweaver-Burk represent seven equally spaced substrate concentrations. The space
between the points increases with the decrease in CS.

The Eadie-Hofstee plot gives slightly better weighting of the data than the Lineweaver-
Burk plot. A disadvantage of this plot is that the rate of reaction r appears in both coordinates
while it is usually regarded as a dependent variable.

Based on the data distribution, the Langmuir plot is the most satisfactory of the three,
since the points are equally spaced.

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 Sample Problems:

1. A series of batch runs with a constant enzyme concentration, the following initial rate
data were obtained as a function of initial substrate concentration.
Cs (mmol/L) 1 2 3 5 7 10 15 20
r, (mmol/L.min) 0.20 0.22 0.30 0.45 0.41 0.50 0.40 0.33

a. Evaluate the Michaelis-Menten kinetic parameters by employing the Langmuir

plot, the Lineweaver-Burk plot, the Eadie-Hofstee plot. In evaluating the kinetic
parameters, do not include data points which deviate systematically from the
Michaelis-Menten model and explain the reason for deviation.
b. Which of the three methods employed gives the best estimate of the kinetic
parameters?

2. Eadie(1942) measured the initial reaction rate of hydrolysis of acetylcholine

(substrate) by dog serum (source of enzyme) and obtained the following data:
Cs (mol/L) 0.0032 0.0049 0.0062 0.0080 0.0095
R, (mol/L.min) 0.111 0.148 0.143 0.166 0.200

Evaluate the Michaelis-Menten kinetic parameters employing (a) the Langmuir plot,
(b) the Lineweaver-Burk plot, and (c) the Eadie-Hofsteeplot.

3. The following data were obtained from enzymatic oxidation of phenol by phenol
oxidase at different phenol concentrations.
Cs (mg/L) 10 20 30 50 60 80 90 110 130 140 150
r (mg/L.min) 5 7.5 10 12.5 13.7 15 15 12.5 9.5 7.5 5.7

a. Determine the constants rmax and KM;

b. Determine the oxidation rate at CS = 70 mg/L.

4. Rates of hydrolysis of p - nitrophenyl – ß – D - glucopyranoside by ß – glucosidase, an

irreversible unimolecular reaction, were measured at several concentrations of the
substrate. The initial reaction rates were obtained as given in the table. Determine
the kinetic parameters of this enzyme reaction.

Cs (mol/m3) r (mol/m3.s)
5.00 4.84x10-4
2.50 3.88x10-4
1.67 3.18x10-4
1.25 2.66x10-4
1.00 2.14x10-4

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 Allosteric Enzymes

Some enzymes have more than one substrate-binding site. The binding of one
substrate to the enzyme facilitates binding of other substrate molecules. This behavior is
known as allostery or cooperative binding, and regulatory enzymes show this behavior. The
rate expression in this case is
𝑟𝑚𝑎𝑥 𝐶𝑆𝑛
𝑟𝑝 =
𝐾𝑚′ + 𝐶𝑆𝑛
Where n = cooperativity coefficient and n>1 indicates positive copperativity. The figure
below shows Michaelis-Menten kinetics with allosteric enzyme kinetics, indicating a
sigmoidal shape. The shape of the curve is different as the increase in the reaction rate is
slower at low substrate concentration due to effectively higher reaction rate.

Shijie Liu, (2013), Bioprocess Engineering, Amsterdam: Elsevier B.V., P. 344

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 Inhibition of Enzyme Reactions

Rates of enzyme reactions are often affected by the presence of various chemicals
and ions.

A modulator or effector is a substance which can combine with enzymes to alter their
catalytic activities. An inhibitor is a modulator which decreases enzyme activity.

Competitive Inhibition

Since a competitive inhibitor has a strong structural resemblance to the substrate,

both the inhibitor and substrate compete for the active site of an enzyme. The
formation of an enzyme-inhibitor complex reduces the amount of enzyme available
for interaction with the substrate and, as a result, the rate reaction decreases. A
competitive inhibitor normally combines reversibly with enzyme. Therefore, the effect
of the inhibitor can be minimized by increasing the substrate concentration, unless
the substrate concentration is greater than the concentration at which the substrate
itself inhibits the reaction.

Digital Image. Wikipedia, 18 May 2020, https://en.wikipedia.org/wiki/Competitive_inhibition

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 The mechanism of competitive inhibition can be expressed as follows:
𝑘1
𝐸 + 𝑆 ↔ 𝐸𝑆
𝑘2
𝑘3
𝐸 + 𝐼 ↔ 𝐸𝐼
𝑘4
𝑘5
𝐸𝑆 → 𝐸 + 𝑃
Utilizing a Michaelis – Menten assumption, the rate equation can be expressed as:
𝒓𝒎𝒂𝒙 𝑪𝒔
𝒓=
𝑲𝒎,𝑰 + 𝑪𝒔
Where:
𝐶
𝐾𝑚,𝐼 = 𝐾𝑚 (1 + 𝐾𝐼 )
𝐼
Km & KI are dissociation constants
𝑘 𝐶 𝐶
𝐾𝑚 = 2 = 𝐸 𝑆
𝑘1 𝐶𝐸𝑆
𝑘4 𝐶𝐸 𝐶𝐼
𝐾𝐼 = 𝑘3
= 𝐶
𝐸𝐼

Therefore, since Km,I is larger than Km, the reaction rate decreases due to the
presence of an inhibitor. It is interesting to note that the maximum reaction rate is not
affected by the presence of a competitive inhibitor. However, a large amount of
substrate is required to reach the maximum rate. The graphical consequences of
competitive inhibition is shown in the figure below.

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 Noncompetitive Inhibition

Noncompetitive inhibitors interact with enzymes in many different ways. They can
bind to the enzymes reversibly or irreversibly at the active site or at some other region.
In any case the resultant complex is inactive.

Digital Image. Wikipedia, 19 April 2020, https://en.wikipedia.org/wiki/Non-competitive_inhibition

The mechanism of noncompetitive inhibition can be expressed as follows:

𝑘1
𝐸 + 𝑆 ↔ 𝐸𝑆
𝑘2
𝑘3
𝐸 + 𝐼 ↔ 𝐸𝐼
𝑘4
𝑘5
𝐸𝐼 + 𝑆 ↔ 𝐸𝐼𝑆
𝑘6
𝑘7
𝐸𝑆 + 𝐼 ↔ 𝐸𝑆𝐼
𝑘8
𝑘9
𝐸𝑆 → 𝐸 + 𝑃

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 Utilizing a Michaelis – Menten assumption, the rate equation can be expressed as:
𝒓𝒎𝒂𝒙,𝑰 𝑪𝒔
𝒓=
𝑲𝒎 + 𝑪𝒔
Where:
𝑟
𝑟𝑚𝑎𝑥,𝐼 = 𝑚𝑎𝑥
𝐶𝐼
1+
𝐾𝐼

Therefore, the maximum reaction rate will be decreased by the presence of a

noncompetitive inhibitor, while the Michaelis – Menten constant Km will not be
affected by the inhibitor. Other reagents need to be added to block binding of the
inhibitor to the enzyme. The comparison of Michaelis-Menten and noncompetitive
inhibition is graphically shown below.

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 Uncompetitive Inhibition

Uncompetitive inhibitors bind to the ES complex only and have no affinity for the
enzyme itself.

The mechanism for this type of inhibition is shown below:

𝑘1
𝐸 + 𝑆 ↔ 𝐸𝑆
𝑘2
𝑘3
𝐸𝑆 + 𝐼 ↔ 𝐸𝑆𝐼
𝑘4
𝑘5
𝐸𝑆 → 𝐸 + 𝑃

Deriving the rate equation,

𝒓𝒎𝒂𝒙,𝑰 𝑪𝒔
𝒓=
𝑲𝒎,𝑰 + 𝑪𝒔
Where:
𝑟 𝐾𝑚
𝑟𝑚𝑎𝑥,𝐼 = 𝑚𝑎𝑥
𝐶𝐼 ; 𝐾𝑚,𝐼 = 𝐶
1+ 1+ 𝐼
𝐾𝐼 𝐾𝐼

The net effect of uncompetitive inhibition is a reduction in both rmax and Km values.
Reduction in rmax has a more pronounced effect than the reduction in Km, and the
net result is a reduction in reaction rate. The graphical consequences of
uncompetitive inhibition is shown in the figure below.

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 Video Guide:
Watch Enzyme Function and Inhibition
Link: https://www.youtube.com/watch?v=PILzvT3spCQ

Sample Problems:

1. Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholine

(substrate) by dog serum (source of enzyme) in the absence and presence of
prostigmine (inhibitor), 1.5x10-7 mol/L obtained the following data:

r (mol/L.min)
Cs (mol/L)
Absence of Prostigmine Presence of Prostigmine
0.0032 0.111 0.059
0.0049 0.148 0.071
0.0062 0.143 0.091
0.0080 0.166 0.111
0.0095 0.200 0.125
a. Evaluate the Michaelis-Menten kinetic parameters in the presence and
absence of prostigmine by employing Langmuir plot, Lineweaver-Burk plot and
Eadie-Hofstee plot;
b. Is prostigmine competitive or noncompetitive?

2. The initial rate of reaction for the enzymatic cleavage of

deoxyguanosinetriphosphate was measured as a function of initial substrate
concentration as follows (Kornberg et al., J. Biol. Chem., 233, 159, 1958):
Cs(μmol/L) r (μmol/L.min)
6.7 0.30
3.5 0.25
1.7 0.16
a. Calculate the Michaelis-Menten constants of the above equation by Langmuir plot,
Lineweaver-Burk plot and Eadie-Hofstee plot;
b. When the inhibitor was added, the initial reaction rate was decreased as follows:
Cs(μmol/L) Inhibitor r (μmol/L.min)
6.7 146 0.11
3.5 146 0.08
1.7 146 0.06

Is this competitive or noncompetitive inhibition? Justify your answer by showing the

effect of the inhibitor graphically employing the three plots. [Contributed by Professor
Gary F. Bennett, The University of Toledo, Toledo, OH]

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 3. The hydrolysis of urea by urease is an only partially understood reaction and shows
inhibition. Data for the hydrolysis of the reaction are given next.
Substrate Concentration 0.2 M 0.02 M
1/r CI 1/r CI
0.22 0 0.68 0
0.33 0.0012 1.02 0.0012
0.51 0.0027 1.50 0.0022
0.76 0.0044 1.83 0.0032
0.88 0.0061 2.04 0.0037
1.10 0.0080 2.72 0.0044
1.15 0.0093 3.46 0.0059
Where r = mol/L.min and CI is inhibitor molar concentration

a. Determine the Michaelis – Menten constant (KM) for this reaction using Lineweaver –
Burk plot.
b. What type of inhibition reaction is this? Substantiate the answer.
c. Based on the answer to part (B), what is the value of KI ?

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 Explain

The activity or performance of an enzyme may be quantified by making use of kinetic

parameters, rmax and Km. Linearized equations from the Michaellis – Menten rate equation
were derived to estimate the values of these kinetic parameters. The linearized plots are
Langmuir, Lineweaver – Burk and Eadie – Hofstee. A series of batch runs with different levels
of substrate concentration should be made and initial reaction rate at each substrate
concentration will be measured. The Cs and r obtained will be substituted to any linearized
plot and values of Km and rmax can be calculated.

The activity of some enzymes can be altered by inhibitory compounds, which bind
the enzyme molecule and reduce its activity. Enzyme inhibition may be competitive,
noncompetitive, and uncompetitive. High substrate and product concentrations may be
inhibitory, too.

Digital Image. Study, https://study.com/academy/lesson/enzyme-inhibitor-definition-examples-quiz.html

Further Reading:

Chapter 8, Section 8.2.4.3, Bioprocess Engineering by Shiejie Liu

(soft copy of the book is uploaded at out google classroom)

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 Elaborate

Formative Assessment:

1. The enzymatic hydrolization of fish oil extracted from crude eel oil has been
carried out using lipase L. One of the desired products is docosa-hexamic acid,
which is used as a medicine in China. For 40 mg of enzyme the Michaelis
constant is 6.2x10-2 (mL/mL) and rmax is 5.6 μmol/mL.min. The balance for fish oil
is then given by
𝑑𝐶𝑠 𝑟𝑚𝑎𝑥 𝐶𝑠%
=−
𝑑𝑡 𝐾𝑚 + 𝐶𝑠%
Where Cs is the fish oil concentration in μmol/mL, and Cs% is fish oil concentration
in mL/mL (vol%). Calculate the time necessary to reduce the concentration of
fish oil from 1.4% to 0.2% vol%. The fish oil density 0.9 g/cm 3 and its molecular
weight is 300.

2. A substrate L – benzoyl arginine p – nitroanilide hydrochloride was hydrolysed by

trypsin, with inhibitor concentrations of 0, 0.3 and 0.6 mmol/L. The hydrolysis rates
obtained are listed in the table below. Determine the inhibition mechanism and the
kinetic parameters (KM , rmax and KI) of this enzyme reaction.
CI (mmol/L)
Cs ( mmol/L) 0 0.3 0.6
0.4 0.79 0.57 0.45
0.15 1.11 0.84 0.66
0.2 1.45 1.06 0.86
0.3 2.00 1.52 1.22

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 Evaluate

Problem Set:

Solve each problem with complete solution and box all final answers.
1. The following data have been obtained for two different initial enzyme
concentrations for an enzyme-catalyzed reaction.
r (CE0 = 0.0015 g/L) in Cs in g/L r (CE0 = 0.00875 g/L) in
g/L.min g/L.min
1.14 20.0 0.67
0.87 10.0 0.51
0.70 6.7 0.41
0.59 5.0 0.34
0.50 4.0 0.29
0.44 3.3
0.39 2.9
0.35 2.5
(a) Find Km; (b) Find rmax for CE0 = 0.015 g/L; (c) Find rmax for CE0 = 0.00875 g/L.
2. An enzyme (cathepsin) hydrolyzes L-glutamyl-L-tyrosine to carbobenzoxy-L-
glutamic acid and L-tyrosine. It has been found (Frantz and Stephenson, BioI.
Chern., 169,359, 1947) that the glutamic acid formed in the hydrolysis, inhibits
(competitively) the progress of the reaction by forming a complex with
cathepsin. The course of the reaction is followed by adding tyrosme
decarboxylase which evolves CO2.
Cs (μmol/mL) CI (μmol/mL) r (μmol/mL.min)
4.7 0 0.0434
4.7 7.57 0.0285
4.7 30.30 0.0133
10.8 0 0.0713
10.8 7.57 0.0512
10.8 30.30 0.0266
30.3 0 0.1111
30.3 7.57 0.0909
30.3 30.30 0.0581
Calculate (a) the value of kinetic parameters of the enzyme, and (b) KI.

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 3. Invertase hydrolyzes cane sugar into glucose and fructose. The following table
shows the amount of sugar inverted in the first 10 minutes of reaction for various
initial substrate concentrations. The amount of invertase was set constant.
Substrate sugar Sugar inverted in 10 min
concentration g/L
g/L
48.9 1.9
67.0 2.1
98.5 2.4
199.1 2.7
299.6 2.5
400.2 2.3
Determine the Michaelis-menten constants of this reaction using Langmuir,
Lineweaver – Burk and Eadie – Hofstee plot.

References

James M. Lee (2008). Fundamentals of Biochemical Engineering. Springer.

Shijie Liu (2013). Bioprocess Engineering. Elsevier B.V.

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