PRT3402- Agricultural Biochemistry
PJJ UPM / UPMET
UNIT 6
Catalysis and regulation of biochemical reactions
Introduction to Unit
Catalysis is the process in which a reaction is speeded-up at rates that
is suitable for the cell machinery to functions. In a biological environment each
reaction is catalysed by the enzymes which are biological catalysts. Within the cell,
there are countless reactions that occur and each reaction is catalysed by a specific
enzyme. The enzymes can be controlled and modulated to suit its specific functions
that respond to the needs and requirement of the cells. In this unit we will learn about
enzyme characteristics, classification, their reaction and kinetics and how their
activities are regulated.
Learning Outcomes
At the end of this unit the students will be able to:
1. Recognise the role, function and classification of enzymes as biological
catalysts.
2. Describe the kinetics of an enzyme reaction and what factors affect an
enzyme reaction.
3. Describe and explain and how it is regulated in response to the cells’
requirement.
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TOPIC 1: Characteristics, classification and kinetics of an enzyme
Main Points
1.1
Most biological catalysts are proteins called enzymes. The substance acted
on by an enzyme is called a substrate. A catalyst increases the rate or
velocity of a chemical reaction without itself being changed in the overall
process. Enzymes speed up reactions by many orders of magnitude. For
example, the enzyme catalase speeds up the conversion of hydrogen
peroxide to water and oxygen by a factor of a billion. True catalysts, such as
enzymes, participate in the reaction, but are unchanged by it. Therefore, they
can continue to catalyze subsequent reaction.
1.2
Enzymes are highly specific for the reactants (substrates) that they act on and
the products that they form. Some exhibit stereosepecificity. The most
important property is that enzymes are reaction specific (does not produce
wasteful byproducts).
1.3
Enzymes can be grouped into regulatory and non-regulatory enzymes. The
regulatory enzymes are usually oligomer and have different sites for substrate
and modulator. Enzymes name are divided into substrate and types of
reactions and added with the term –ase at the end, their historical name or
named after their genes (Rec A name after rec A gene). The Enzyme
Commission (EC) of the International Union of Biochemistry and Molecular
Biology (IUBMB) classed enzymes into 6 groups based on their chemical
reactions. All enzymes fit into one of six major classes based on the type of
reaction catalysed.
1.4
The six classes of reactions are as follows:
1. OXIDOREDUCTASES
Catalyse oxidation-reduction. Most of these
enzymes are referred to as dehydrogenases, but some are called
oxidases, peroxidase, oxygenases or reductases.
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2. TRANSFERASES. Catalyse group-tranSfer reactions and many require
the presence of coenzymes. A portion of a substrate molecule usually
binds covalently to the enzyme or its coenzyme. This group includes
kinases, enzymes that catalyze the transfer of phosphoryl group from ATP.
3. HYDROLASES Catalyse hydrolysis. They are special class of
transferases, with water serving as the acceptor of the group transferred.
4. LYASES Catalyse lysis of a substrate, generating a double bond; these
are non-hydrolytic, non-oxidative elimination reactions. In the reverse
direction, lyases catalyse the addition of one substrate to a double bond of
a second substrate. A lyase that catalyses an addition reaction in cells is
often termed a synthase.
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5. ISOMERASES. Catalyse structural change within a single molecule
(isomerisation reaction). These reactions have only one substrate and one
product (simplest enzymatic reaction).
6. LIGASES. Catalyse ligation or joining of two substrates. These reaction
require the input of the chemical potential energy of a nucleoside
triphosphate such as ligase ATP.
Ligases are usually referred to as
synthetases.
1.5
All enzymes will have their unique EC number. For example the enzyme
ATP:glucosephosphotransferase (EC 2.7.1.1). The first number is the class 2belonging to transferase group of reaction, the second is subclass
7-
transfers phosphate), the third number is subsubclas 1- alcohol as acceptor of
phosphate and the final 1 is the serial number for the enzyme in its subclass.
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1.6
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For example EC 5.3.1.1 is for triose phosphate isomerase. The first three
numbers are the class, sub-class and sub-subclass. The last is the serial
number for the enzyme in its subclass.
1.7
Enzyme activity can be regulated, varying in response to the concentration of
substrates or other molecules. Nearly all enzymes are proteins, although a
few catalytically active RNA molecules have been identified.
1.8
Catalysts change the rates of reactions, but do not affect the equilibrium of a
reaction. That is, you cannot make more product from an enzyme-catalyzed
reaction than you can from the same reaction without it. The enzyme simply
helps to reach the equilibrium state faster than if it were not present.
1.9
The enzyme or catalyst works simply by lowering the energy barrier of a
reaction, the diagram below showed the energy barrier for an un-catalysed
and catalysed reaction. By doing so, the catalyst increases the fraction of
molecules that have enough energy to attain the transition state, thus making
the reaction go faster in both directions. The position of the equilibrium (the
amount of product versus reactant) is unchanged by a catalyst.
1.10
The active site of an enzyme is the region that binds the substrate and
converts it into product. It is usually a relatively small part of the whole
enzyme molecule and is a three dimensional entity formed by amino acid
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residues that can lie far apart in the linear polypeptide chain. The active site is
often a cleft or crevice on the surface of the enzyme that forms predominantly
non-polar environment which enhances the binding of the substrate.
1.11
The substrate(s) is bound in the active site by multiple weak forces
(electrostatic interactions, hydrogen bonds, van der Waals bonds and
hydrophobic interactions) and in some cases by reversible covalent bonds.
When the substrate has bound to the active site, an enzyme-substrate
complex is formed. Catalytic active residues within the active site of the
enzyme act on the substrate molecule to transform it first into the transition
state complex and then into product, which is released into solution. The
enzyme is then free to bind another molecule of substrate and begin its
catalytic cycle again.
1.12
Enzymes bind substrate transiently (short time). The lock-and-key model
proposes that an enzyme/substrate pair is like a lock and key. Though it
explains the specificity of enzyme/substrate pairs, it does little to explain
catalysis, because a lock does not change a key the way an enzyme changes
a substrate. In 1958, Daniel Koshland proposed the induced fit model to
explain enzymatic catalysis. The model proposes that distortion of the enzyme
and the substrate is an important event in catalysis
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1.13
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Enzymes do more than simply bind and position substrates. Enzymes bind
substrate(s); lower the energy of the transition state; and directly promote the
catalytic event. The properties and spatial arrangement of the amino acid
residues forming the active site of an enzyme will determine which molecules
can bind and be substrates for the enzyme. Substrate specificity is often
determined by changes in relatively few amino acids in the active site.
1.14
The enzyme (E) binds the substrate to form an enzyme-substrate complex
(ES). It is given by the general formulation as follows: E + S  ES  P. The
substrate reacts transiently with the protein catalyst to form the product. The
rate of an enzymatic reaction depends on the concentration of both substrate
and enzyme. At saturating concentration of substrate the reaction is first
order. The more enzyme the faster the reaction.
1.15
The rate of an enzyme catalysed reaction is called velocity. Initial velocity V0
(μmol min-1) is the rate where the product is not yet present. A plot of product
formed against time for an enzyme-catalysed reaction shows an initial period
of rapid product formation which gives a linear portion of the plot.
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1.16
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This is followed by a slowing down of the enzyme rate as substrate is used up
and/or as enzyme loses activity. V0 is obtained by drawing a straight line
through the linear part of the curve, starting at the zero time-point. The slope
of this straight line is equal to V0. The most common way to express enzyme
activity is the initial rate (V0) of the reaction being catalysed. The unit is μmol
min-1 (μmol of substrate transformed per minute).
1.17
At low substrate concentration doubling of [S] will lead to a doubling of V0.
However at higher substrate concentration the enzyme becomes saturated
and further increases in [S] leads to a small change in V0.This occurs at
saturating substrate concentration where effectively all of the enzymes
molecules have bound substrate. The overall enzyme rate is now dependant
on the rate at which the product can dissociate from the enzyme, and adding
further substrate will not affect this. The shape of the resulting graph when V0
is plotted against [S] is called a hyperbolic curve (Figure below)
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1.18
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When the substrate concentration is saturating (i.e. all the enzyme molecules
are bound to substrate), a doubling of the enzyme concentration will lead to a
doubling of V0.
1.19
The Michaelis-Menten Model uses the following concept of enzyme catalysis:
k1
E+S
k3
ES
E+P
k2
The enzyme (E) combines with its Substrate (S) to form an enzyme-substrate
complex (ES). The ES complex can dissociate again to form E + S, or it can
proceed chemically to form E and the product P. The rate constant k1, k2, and
k3 describe the rates for each step of the catalytic process. It is assumed
there is no significant rate for the backward reaction of enzyme and product
(E + P) being converted to ES complex. [ES] remains approximately constant
until all the substrate is used, hence the rate of synthesis of ES equal its rate
of consumption over most of the course of the reaction, that is, [ES] maintains
a steady state.
1.20
From the observation of the properties of many enzymes it was known that
the initial velocity (V0) at low substrate concentration is directly proportional to
[S].
While at high substrate concentrations the velocity tends towards
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maximum value, that is, the rates become independent of [S]. This maximum
velocity is called Vmax (μmol min-1). The initial velocity V0 is the velocity
measured experimentally before than 10% of the substrate has been
converted to product. Michaelis-Menten derived an equation to describe this
observation. The Michaelis –Menten equation is
V0 = Vmax [S]
Km + [S]
The equation describes a hyperbolic curve as shown below.
1.21
In deriving the equation, Michelis and Menten defined a new constant, Km,
the Michaelis constant [Molar (i.e. per mole), M]
Km = k2 + k3
k1
1.22
Km is a measure of the stability of the ES complex, being equal to the sum of
the rates of breakdown of
ES over its formation. For many enzymes k2 is
much greater than k3. Under these circumstances Km becomes a measure of
the affinity of the enzyme for its substrate. A high Km indicates a weak
substrate binding (k2 predominant over k1), and low Km indicates a strong
substrate binding (k1 predominant over k2). Km can be measured
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experimentally because Km is equivalent to the substrate concentration at
which the velocity is equal to half of Vmax.
1.23
The Lineweaver-Burk plot is a plot of 1/V0 against 1/[S]. This plot is a
derivation of Michaelis –Menten equation:
1 =
1
V0
Vmax
+
Km
1
Vmax
[S]
which gives a straight line, with the intercept on the y-axis equal to 1/Vmax and
intercept on the x-axis equal to -1/Km. The slope of the line is equal to Km /
Vmax. The Lineweaver is useful in determining how an inhibitor binds to an
enzyme.Although many enzymes conforms to Michaelis –Menten kinetics, a
few enzymes called allosteric enzymes do not.
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TOPIC 2 : Enzyme inhibition and regulation
Main Points
2.1
Any molecule which acts directly on an enzyme to lower its catalytic activity
is called an inhibitor. Some enzyme inhibitors are normal body metabolites that
inhibit a particular enzyme as part of the normal metabolic control of a pathway.
Other inhibitors may be foreign substances, such as drugs or toxins, where the
effect of enzyme inhibition could either be therapeutic or, at the other extreme,
lethal. Enzyme inhibition is of two main types: irreversible or reversible.
Reversible inhibition is subdivided into competitive and noncompetitive inhibition.
2.2
Irreversible inhibition occurs when substances combine covalently with
enzymes so as to inactivate them irreversibly. The inhibitors often form covalent
bond to an amino acid residue at or near the active sited, and permanently
inactivate the enzyme. Susceptible amino acid residues include Ser and Cys
residues which have reactive –OH and –SH groups, respectively. Almost all
irreversible enzyme inhibitors are toxic substances, either natural or synthetic.
Some examples are cyanide and penicillin.
2.3
In reversible inhibitive competition, the competitive inhibitor typically has
close structural similarities to the normal substrate of the enzyme. Thus it
competes with substrate molecules to bind to the active site. The enzyme may
bind either a substrate molecule or an inhibitor molecule, but not both at the
same time. The competitive inhibitor binds reversibly to the active site. At high
substrate concentrations the action of a competitive inhibitor is overcome
because a sufficiently high substrate concentration will successfully compete out
the inhibitor molecule in binding to the active site.
2.4
In reversible non-competitive inhibition, a non-competitive inhibitor binds to a
site other than the active site and causes a change in the overall 3D-structure of
the enzyme that leads to a decrease in catalytic activity. The enzyme may bind
substrate, the inhibitor or both substrate and inhibibitor. The effects of noncompetitive inhibitor cannot be overcome by increasing the substrate
concentration, so there is a decrease in Vmax. The affinity of the enzyme for the
substrate is unchanged and so Km remains the same.
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2.5
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The following table showed the effects of inhibitors on the kinetic constants.
Type of inhibitor
Effect on kinetic constants
Competitive (I binds to E only)
Raises Km
Vmax remains unchanged
Noncompetitive (I binds to E or ES)
Lowers Vmax
Km remains unchanged
Uncompetitive (I binds to ES only)
Lowers Vmax and Km
Ratio of Vmax/Km remains
unchanged
2.6
The following diagrams depict the mechanisms of inhibition described earlier.
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2.6
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Regulatory enzymes are classified by the method of their modulation.

Allosteric modulation

Covalent modification
Allosteric enzymes modulation - These enzymes are often multi-subunit proteins,
with one or more active site on each subunit. The binding of substrate at one
active site induces conformational change in the protein that is conveyed to the
other active sites, altering their affinity for substrate molecules. In addition,
allosteric enzymes may be controlled by effector molecules (activators and
inhibitors) that bind to the enzyme at a site other than the active site (either on
the same subunit or on a different subunit). Binding of the activator or inhibitor
causes a change in the conformation of the active site which alters the rate of
enzyme activity (e.g. the binding of CO2 and H+ to haemoglobin). An allosteric
activator increases the rate of enzyme activity while an allosteric inhibitor
decreases the activity of the enzyme.
2.8
Covalent modification activates some enzymes and inactivates others. One of
the most widespread modifications is phosphorylation or dephosphorylation of
various amino acid side chains (e.g., serine, threonine, tyrosine, and histidine).
These kinds of modification are most often a part of complex regulatory
pathways, frequently under hormonal control. Another example of covalent
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enzyme activation is proteolytic cleavage, found in the pancreatic proteases
(such as trypsin, chymotrypsin, elastase, and carboxypeptidase). These enzymes
are synthesized in the pancreas as a slightly longer, catalytically inactive
molecules called zymogens (trypsinogen, chymotrypsinogen, proelastase, and
procarboxypeptidase,
respectively).
The
zymogens
proteolytically in the intestine to yield the active enzymes.
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must
be
cleaved