Biochemical Engineering by Mary Rose F.

Persincula

CHAPTER 2:
ENZYME AND ENZYME KINETICS

Objectives:
1. To define what enzyme is.
2. Give the classification of enzyme and the nomenclature for which.
3. Show the two approaches in deriving the enzyme kinetic equation.
4. Describe the enzyme kinetic equation by applying the different plot schemes used
for obtaining maximum velocity and specific rate of reaction of Micheali’s constant.
5. Describe the inhibition of enzyme activity and the manner of regulation of enzyme
activity.
6. Analyze the impact of pH, temperature and other factors affecting enzyme kinetics
and activity.

1. ENZYMES

Enzymes are proteins produced by living cells which catalyze biochemical reactions
necessary for the functioning of cells. Enzymes have high molecular weight (15,000 < MW <
several million Daltons). Holoenzyme is an enzyme that contains non-protein group called
cofactors such as metal ions: Mg, Zn, Mn, and Fe; or coenzyme such as a complex organic
molecules: NAD or vitamins. Apoenzyme is the protein part of the holoenzyme.

Holoenzyme = Apoenzyme + Cofactor (Coenzyme) (1)

The biomolecules which are acted upon by enzymes are collectively termed substrates.
Enzymes are highly specific to target substrates, and their specificity and activity are related
to their protein structure (Lee, 1992).

An active site of the enzyme molecule, often due to its molecular conformation, is the region
which targets a specific biochemical reaction. Here, physical and chemical interactions with
the substrate lead to the catalysis of the chemical reaction.

9
Biochemical Engineering by Mary Rose F. Persincula

In biochemical engineering, enzymes are produced by fermentation for industrial, medical

and analytical uses, or are utilized in bioreactors (in solution or immobilized) for the catalytic
production of other useful compounds.

1.1. Enzyme Classification and Nomenclature

Enzyme is named by adding the suffix –ase to the end of the substrate name that is to be
converted to the desired product or to the reaction which is catalyzed. Example: (1) urease
that changes urea into ammonium carbonate, (2) protease that converts protein or
polypeptides to smaller molecules such as amino acids; and, (3) phosphoglucose isomerase
that converts glucose-6-phosphate to fructose-6-phosphate, (4) alcohol dehydrogenase that
catalyzes the removal of hydrogen from alcohol.

Since 1926, more than 3000 enzymes are identified. The Commission on Enzymes of the
International Union of Biochemistry classified enzymes last 1864 into six major classes as
shown in Table 6 (Refer to Enzyme Nomenclature, 1992, Academic Press, San Diego,
California, ISBN 0-12-227164-5). Each of the major classes is further divided into numerical
subclasses and sub-subclasses depending on the reaction involved. Examples of enzymes
with their enzyme classification numbers proposed by the Enzyme Commission (EC) are
shown in Table 6.

Table 6. Major Enzyme Classes According to the Enzyme Commission.

(from B. Atkinson and F. Mavituna. 1991. Biochemical
Engineering and Biotechnology
Ltd., Hampshire, U.K. p. 522)
Major class number Class
1 Oxidoreductases
2 Transferases
3 Hydrolases
4 Lyases
Handbook, Macmillan Publishers,
Reactions catalyzed
Oxidation-reduction
Functional group transfer (from donor to
acceptor)
Hydrolysis
Removal of group, leaving double bond
Addition of group to double bond (forming new
double bond or a new ring structure)

10
Biochemical Engineering by Mary Rose F. Persincula

5 Isomerases Isomerization
6 Ligases Joining of two molecules coupled with cleavage
(synthetases) of a pyrophosphate bond of ATP or similar
triphosphate

Table 7. Examples of Enzymes With Their Classification Numbers.

(from B. Atkinson and F. Mavituna, 1991. Biochemical Engineering
and Biotechnology Handbook, Macmillan Publishers, Ltd.,
Hampshire, U.K. p. 522 - 523)
EC number Systematic name Trivial name
Oxidoreductases
1.1.1.1 Alcohol:NAD Alcohol dehydrogenase
oxidoreductase
1.11.1.6 H2O2: H2O2 Catalase
oxidoreductasse
Transferases
2.1.1.6 S-adenosylmethionine Catecholmethyltransferase
catechol
Hydrolases
3.2.1.1 α-1,4-Glucan α-amylase
glucanohydrolase
3.2.1.2 α-1,4-Glucan β-amylase
maltohydrolase
3.2.1.4 β-1,4-Glucan Cellulose
glucanohydrolase
Lyases
4.1.3.6 Citrate oxaloacetate Citrate lyase, citrase
Isomerases
5.3.1.5 D-Xylose ketol isomerase Xylose isomerase, glucose
isomerase
Ligases
6.2.1.1 Acetate:CoA ligase Acetyl-coA synthetase
6.4.1.1 Pyruvate:CO2 ligase Pyruvate carboxylase
Due to the complexity of the classification system difficulties are often experienced in

11
Biochemical Engineering by Mary Rose F. Persincula

accurately describing the reaction involved. This is compounded by the fact that a trivial
enzyme nomenclature was already well established before the introduction of the
international system. Table 7 shows some enzyme categories used in the trivial enzyme
classification.

Table 8. Some Enzyme Categories Used in the Trivial Enzyme Classification.

(from B. Atkinson and F. Mavituna, 1991. Biochemical
Engineering and Biotechnology Handbook, Macmillan Publishers,
Ltd., Hampshire, U.K. p. 524)
Name Reaction catalyzed

Dehydrogenases dehyrogenation of substrate with hydrogen transfer to a molecule

other than molecular oxygen
Hydroxylases hydroxylation of substrate with molecular oxygen as donor
Kinases transfer of phosphate from nucleoside triphosphate to substrate
Mutases migration of phosphate group from one position to another within the
same molecule
Oxidases oxidation of substrate with molecular oxygen as donor
Oxygenases incorporation of oxygen molecule into substrate with oxidative
cleavage of a carbon-carbon bond
Phosphatases hydrolysis of phosphate ester
Phosphorylases addition of elements of phosphoric acid across glycosyl or related
bond
Synthetases condensation of two molecules cleavage of nucleoside triphosphate
Thiokinases formation of thiol ester from carboxylic acid substrate with cleavage
of ATP
Transferases transfer of group from one substrate to another

2. ENZYME CATALYSIS

Enzyme acts as a catalyst which increases the rate of a chemical reaction without itself being
consumed or transformed. It participates in reactions but is neither a chemical reactant nor a
chemical product. It provides an alternative pathway of lower activation energy for a reaction

12
Biochemical Engineering by Mary Rose F. Persincula

to proceed while remaining at equilibrium or no permanent change on the catalyst themselves

by binding the substrate and forming a substrate-enzyme complex which produces the desired
product. It lowers the activation energy of the catalyzed reaction, but does not affect free
energy change or equilibrium constant.

Figure 4. Activation energy for catalyzed and uncatalyzed reactions.

Free energy (G) is the energy stored in the bonds of a chemical that can be harnessed to do
work. Free energy change (ΔG) of a reaction refers to the change between the free energy in
the product(s) and that in the substrate (s). The free energy change determines the reaction
equilibrium – the maximum amount of the product that could be theoretically produced.
Reaction equilibrium is represented by reaction equilibrium constant

Keq,=γp[P]/ γs[S] (2)

- ΔG, uncatalyzed=RTln Keq (3)

[] represents the concentration of the compounds.

γp and γs are activity constants of the product and the substrate, respectively.

The catalyst activity increases the amount of the product at reaction equilibrium and
accelerates the reaction rate to reach the reaction equilibrium.

3. Enzyme Kinetics

3.1. Simple Enzyme Kinetics

Numerous studies have shown that the substrate binds to a specific region of the enzyme

13
Biochemical Engineering by Mary Rose F. Persincula

(called the active site) to form an enzyme-substrate complex (Bailey and Ollis1986).
Catalysis is achieved by the formation of the enzyme-substrate complex, which lowers the
activation of the reaction.

The mechanistic models for simple enzyme kinetics (i.e., single-substrate single-enzyme) is
based on the formation of the enzyme-substrate complex, and followed by a dissociation step
of the complex to form the product and free enzyme. Schematically, the reactions are
represented as,

⇄k1
(1)
ES ES 
k2
EP
k 1

where E is the free enzyme, S is the substrate, ES is the enzyme-substrate complex, and P is
the product. The rate expressions for the enzyme catalyzed reactions can be derived from
either of two approaches: (1) the rapid equilibrium assumption (Michaelis-Menten approach),
which assumes the product releasing step (k 2) is the rate limiting step while the reversible
reaction (k1, k-1) is at equilibrium, or by (2) the quasi-steady state assumption (Briggs and
Haldane approach), which assumes that the rate of change of the intermediate enzyme-
d  ES 
substrate complex is negligible (i.e., 0 ) . Both approaches yield the same
dt
expression and differ only in the interpretation of the constant, Km. If the rate of product
dP
formation is represented by v  , the final form of the rate expression for this simple
dt
type of enzyme kinetics is:

v max  S 
v (2)
Km   S 

where [S] is the substrate concentration, vmax is the maximum reaction rate and Km is a
constant. In the Michaelis-Menten approach, Km is commonly known as the Michaeli’s
constant, which is equal to k-1/k1. In the Briggs and Haldane approach, Km is interpreted as
[(k-1+ k2)/k1]. Both will be the same if the product releasing step is slower than the
dissociation of the enzyme-substrate complex (i.e. k2<<k-1). In many biochemical engineering
literature, equation (2) is often known as the Michaelis-Menten equation.

14
Biochemical Engineering by Mary Rose F. Persincula

Equation (2) is representative of a saturation type kinetics (i.e., the reaction rate achieves a
maximum saturated value at high reactant concentrations).

vmax

½ vmax
Reaction velocity, v

[S]=Km

Substrate concentration [S]

Figure 4. Dependence of Enzyme Reaction Rate on Substrate Concentration.

2.2. Michaelis-Menten Type Kinetics

Linearized plots of the Michaelis-Menten equation have been used to evaluate the values of
the constants vmax and Km.

Table 9. Linearized Plots of the Michaelis-Menten Equation.

Name of Equation Slope Intercept Plot
linearized plot

Lineweaver- 1 Km 1 1 Km 1
  1
Burk plot v v max [ s ] v max v max v max v
1
v max
1 1

Km S

15
Biochemical Engineering by Mary Rose F. Persincula

Eadie-Hofstee 1  Km v max v max

v   Km  v max v
plot [S ]

v max
Km
v
[S ]

Hanes-Woolf [S ] 1 Km 1 Km
 [S ]  [ s]
plot v v max v max v max v max v
Km
v max
 Km [S ]

2.3. Inhibited Enzyme Kinetics

Enzyme inhibitors are compounds which bind to enzymes and reduce their activity, and may
either be reversible or irreversible (Shuler, 2002). There are three types of reversible enzyme
inhibitions: competitive, noncompetitive, and uncompetitive. In some cases, the substrate
may also act as inhibitors.

Table 10. Types of Reversible Enzyme Inhibition.

Inhibition type Double reciprocal (Lineweaver Relevant equations and
Burke) plot features
Competitive inhibition v
v max[ S ]
1/ v I>0
inhibitor is usually a Km, app  [ S ]
I=0
substrate analog which
1/ vmax where
competes for the active site [I ]
Km, app  Km(1  )
of the enzyme Ki
-1/ Km -1/ Km, app
1/ [S] - increased value of
Km, app
- vmax not affected

16
Biochemical Engineering by Mary Rose F. Persincula

Noncompetitive inhibition v max, app

1/ v v
inhibitor is not a substrate I>0  Km 
1  
I=0  [ S ] 
analog but bind on sites
1/ v max app where
other than the active site
v max
which reduces affinity of 1/ v max v max, app 
 [I ] 
1  
enzyme to substrate  Ki 
-1/ Km
1/ [S] -reduction in vmax
- Km remains unchanged
Uncompetitive inhibition v max, app [ S ]
1/ v
v
I>0 Km, app  [ S ]
inhibitor binds to the ES
I=0 where
complex only and has no 1/ v max app
v max, app, Km, app are
affinity for the enzyme similarly defined above
1/ v max
itself
-1/ Km, app -1/ Km 1/ [S]

Substrate inhibition occurs when when high substrate concentrations decreases the activity of
the enzyme. It is shown graphically below:

1/ v With substrate
inhibition
no substrate
inhibition

1/ v max

-1/ Km 1/ [S]

Figure 5. Lineweaver-Burke Plots of a System With Substrate Inhibition.

17
Biochemical Engineering by Mary Rose F. Persincula

2.4. Models for more Complex Enzyme Kinetics

Table 11. Different Models Which May be Used to Predict the Specific Growth Rate (Shuler,
2002).
Equation
Substrate Monod S
 m (3)
limited Ks  S
growth Blackman   m , iff S  2 Ks
 (4)
  m S iff S  2 Ks
2 Ks
Moser mSn  n 1
  m(1  KsS )
Ks  Sn
(5)

Contois mS
 (6)
Ksx  S
Substrate Non-competitive mS

inhibited substrate  Ks  S  (7)
1  1  
growth inhibition  S  Ki 

18
Biochemical Engineering by Mary Rose F. Persincula

Competitive mS

substrate  S  (8)
Ks 1    S
inhibition  Ki 
Product Non-competitive mS

inhibited product  Ks  P 
1  1  
S  Kp 
(9)
growth inhibition 

Competitive mS

product  P 
Ks1    S (10)
inhibition  Kp 

19