IBS PROGRAMMEMBBS 200 level
ENZYMOLOGY 1
outline
• Introduction
• Basic concept of enzymes
• Classification of enzymes
• Properties of Enzymes
• Nature of enzymes
• Factors affecting enzyme action
• Assignments
Introduction
• Kinetics is the study of the rates at which
chemical reactions occur
• Enzyme kinetic is a branch of chemical kinetic
and shares much same features
• Enzymes are biologic polymers that catalyze
the chemical reactions that make life as we
know it possible
Introduction cont’d
• The presence and maintenance of a complete
and balanced set of enzymes is essential for
the breakdown of nutrients to supply energy
and chemical building blocks into DNA,
proteins, membranes, cells and tissues and
the generation of energy to power cell
motility, neural function & muscle contraction
CLASSIFICATION OF ENZYMES
Figure 1. Six major classes of Enzymes
Classification with examples
• Class 1: Oxidoreductases : enzymes will catalyse
oxidation of one substrate with another substrate or
co-enzyme
AH₂ + B -------------→ A + BH₂
for example,
• Alcohol + NAD⁺ ----→ Aldehyde + NADH + H⁺
• The enzyme is Alcohol dehydrogenase;
• Systemic name is Alcohol-NAD-oxidoreductase;
• Oxidoreductases may also oxidize substrates by
adding oxygen, e.g. oxidases, oxygenases and
dehydrogenases with simultaneous reduction of
substrates
Classification with examples cont’d
Class 2:Transferases:
• enzymes transfers one group(other than
hydrogen) from the substrate to another
substrate.
• This may be represented as
• A-R + B → A + B-R ,
• For example,
• Hexose + ATP → Hexose-6-phosphate + ADP
• The name of enzyme is Hexokinase
Classification with examples cont’d
Class 3: Hydrolases
• This class of enzymes can hydrolyse ester, ether,
peptide or glycosidic bonds by adding water and
then breaking the bond.
• Acetyl choline + H₂O --------→ Choline + acetate
• The enzyme is Acetylcholine esterase or
• Acetyl choline hydrolase (systematic)
• All digestive enzymes are hydrolases-found in
the lysosomes
Classification with examples cont’d
Class 4: Lyases:
• these enzymes can remove groups from
substrates or break bonds by mechanisms other
than hydrolysis.
• For example:
• Fructose 1,6-bisphosphate -----→
Glyceraldehyde-3-phosphate
+ dihydroxy acetone phosphate
• The enzyme is aldolases
Classification with examples cont’d
Class 5: Isomerases
• These enzymes can produce optical, geometric
or positional isomers of substrates.
Examples are :
• Racemases, epimerases, cis-trans isomerases.
• Glyceraldehyde-3-phosphate →
Dihydroxy-acetone-phosphate
• Enzyme is Triose phosphate isomerase
Classification with examples cont’d
Class 6: Ligases
• Enzymes link two substrates together, these
usually with the simultaneous hydrolysis of ATP,
• (Latin, Ligare = to bind).
• For example:
• Acetyl CoA + CO₂ + ATP → Malonyl CoA + ADP + Pi
• Enzyme is Acetyl CoA carboxylase.
Classification with examples cont’d
Thermodynamic consideration
• From the standpoint of energy, the enzymatic
reactions are divided into 3 types:
1. Exergonic or Exothermic Reaction
Here energy is released from the reaction and
therefore reaction essentially goes to completion,
e.g. urease enzyme
• Urea → ammonia + CO₂ + energy
• At equilibrium of this reaction, the substrate will be
only 0.5% and product will be 99.5%.
• Such reactions are generally irreversible
Thermodynamic consideration cont’d
2. Isothermic Reaction
• When energy exchange is negligible, and the reaction
is easily reversible, e.g.
• Pyruvate + 2H⁺ ↔ Lactate
3. Endergonic or endothermic reaction
• Energy is consumed and external energy is to be
supplied
for these reactions
• In the body this is usually accomplished by coupling
the endergonic reaction with an exergonic reaction,
e.g. Hexokinase catalyses the following reaction:
• Glucose + ATP → Glucose-6-phosphate + ADP
Properties of Enzymes
Properties of Enzymes
• Enzymes are protein catalysts that increase the
velocity of a chemical reaction and are not consumed
during the reaction
• NB-some RNA act like enzymes, usually catalyzing the
cleavage and synthesis of phosphodiester bonds
• RNA with catalytic activity are called ribozymes
• They are labile
• They are water-soluble
• They can be precipitated by protein precipitating
reagents (ammonium sulfate or trichloroacetic acid)
• They contain 16% weight as nitrogen
Properties of Enzymes cont’d
1).Holoenzymes -The catalytic activity of many
enzymes depends on the presence of small molecules
termed cofactors, although the precise role varies with
the cofactor and the enzyme.
• Such an enzyme without its cofactor(i.e protein
portion) is referred to as an apoenzyme;
• the complete, catalytically active enzyme is called a
holoenzyme.
• Apoenzyme + Cofactor= Holoenzyme
• Cofactors can be subdivided into two groups:
metals(metalloenzymes) and small organic
molecules (coenzymes).
Properties of Enzymes cont’d
• The enzyme carbonic anhydrase, for example,
requires Zn²⁺ for its activity .
• Glycogen phosphorylase which mobilizes glycogen
for energy, requires the small organic molecule
pyridoxal phosphate (PLP)cofactors that are small
organic molecules are called coenzymes
• Coenzymes are often derived from vitamins, can
either be tightly or loosely bound to the enzyme
• If tightly bound, they are called prosthetic
.
groups(e.g FAD) .
• Coenzyme NAD⁺ contains niacin & FAD contain
riboflavin
Some Enzymes in relation to coenzymes
Coenzymes Sources Enzymes
Thymine pyrophosphate (TPP) Thiamin Pyruvate dehydrogenase
Flavine adenine nucleotide Riboflavin Monoamine oxidase
Nicotinamide adenine Niacin Lactate dehydrogenase
dinucleotide (NAD⁺)
Pyridoxal phosphate (PLP) Pyridoxine Glycogen phosphorylase
Coenzyme A Pantothenic acid Acetyl CoA carboxylase
Biotin Pyruvate carboxylase
5’-Deoxyadenosyl cobalamine cyanocobalamin, Methylmalonyl mutase
Tetrahydrofolate Folate Thymidylate synthase
S-adenosylmethionine methionine Methionine synthase
Enzyme metals Cofactor:Metalloenzymes
Metals Enzymes
Zn²⁺ Carbonic anhydrase, ALA Dehydratase
Zn²⁺ Carboxypeptidases
Mg²⁺ Hexokinase
Ni ²⁺ Urase
Mo²⁺ Nitrate reductase
Se ²⁺ Glutathione peroxidase
Mn²⁺ Superoxide dismutase
K⁺ Propyonyl CoA carboxylase
(Fe-Cu)²⁺ Tyrosine hydroxylase
Properties of Enzymes cont’d
2). Transformation of energy
• In many biochemical reactions, the energy of the
reactants is converted with high efficiency into a
different form
• In mitochondria, the free energy contained in small
molecules derived from food is converted first into
the free energy of an ion gradient and then into a
different currency, the free energy of adenosine
triphosphate ATP
• Enzymes may then use the chemical-bond energy of
ATP in many ways
• The enzyme myosin converts the energy of ATP into
the mechanical energy of contracting muscles
Properties of Enzymes cont’d
3).Catalytic efficiency
• Enzyme catalyzed reaction are highly efficient , proceeding
from 10ᶟ-10⁸ times faster than uncatalyzed reactions
• the no of molecules of substrate converted to product per
enzyme molecule per second is called the turnover
number or Kcat and typically is 10²-10⁴/s
4).Specificity-enzymes are highly specific ,interacting with
one or a few substrate and catalyzing only one type of
chemical reaction .
• Enzymes are extremely selective catalysts, both to the
type of reaction catalyzed and for a single substrate or a
small set of closely related substrates
• Note :set of enzymes made in a cell determines which
metabolic pathway occur in that cell
Figure 2: Enzyme specificity.
A- Trypsin cleaves on the carboxyl side of arginine & lysine residues
B-thrombin cleaves Arg-Gly bonds in particular sequence specifically
Properties of Enzymes cont’d
5). Capacity to control : the catalytic activity of many
enzymes vary in response to the concentrations of
substances other than their substrates and products. The
mechanisms of these regulatory processes include
allosteric control, covalent modification of enzymes and
variation of the amounts of enzyme synthesized
6). Location within the cell: many enzymes are localized
in specific organelles within the cell, such
compartmentalization serves to isolate the reaction
substrate or products from other competing reaction, this
provides a favourable environment for the reaction and
organizes the thousands of enzymes present in the cell
Mitochondria
TCA cycle
Fatty acid oxidation
Oxidation of pyruvate
Lysosome Peroxisomes Cytosol
Degradation of complex catalase .Glycolysis
macromolecules, H₂O₂,VLCFA .HMP pathway
digestive enzymes .Fatty acid synthesis
Nucleus
DNA and RNA synthesis
Figure 3: The intracellular location of some important Biochemical pathways
Nature of Enzymes
Nature of Enzymes
A. Active site: enzyme molecules contain a special
pocket or cleft called the active site-contain amino
acid side chain that participate in substrate binding
and catalysis.
• Glutathione GSH helps keep redox-sensitive active
sites on enzyme in the necessary reduced state
• The substrate bind the enzyme, forming an enzyme-
substrate(ES) complex
• Binding is thought to cause conformational change in
the enzyme(induced fit) that allows catalysis
• (E )+ (S)↔ ES-complex ↔ E-P complexE + P
• E=enzyme; S=subtrate; P=product
B. Substrate Binding Sites
• Enzyme specificity (the enzyme’s ability to react
with just one substrate) results from the three-
dimensional arrangement of specific amino acid
residues in the enzyme that form binding sites
for the substrates and activate the substrates
during the course of the reaction.
• The “lock-and-key” and the “induced-fit”
models for substrate binding describe two
aspects of the binding interaction between the
enzyme and substrate.
1. Lock-and-key model for substrate binding
• The substrate binding site contains amino acid
residues arranged in a complementary three-
dimensional surface that “recognizes” the substrate
and binds it through multiple hydrophobic
interactions, electrostatic interactions, or hydrogen
bonds
• The amino acid residues that bind the substrate can
come from very different parts of the linear amino
acid sequence of the enzyme, as seen in glucokinase.
• In the lock-and-key model, the complementarity
between the substrate and its binding site is
compared to that of a key fitting into a rigid lock
2. “Induced fit” model for substrate binding
• As the substrate binds, enzymes undergo a
conformational change (“induced fit”) that repositions
the side chains of the amino acids in the active site and
increases the number of binding interactions
• The induced fit model for substrate binding recognizes
that the substrate binding site is not a rigid “lock” but
rather a dynamic surface created by the flexible overall
three-dimensional structure of the enzyme.
• The function of the conformational change induced by
substrate binding, the induced fit, is usually to
reposition functional groups in the active site in a way
that promotes the reaction, improves the binding site of
a cosubstrate, or activates an adjacent subunit through
cooperativity
Figure 4. Reaction in the enzyme active catalytic site.
• The multiple interactions between the substrate and
the enzyme in the catalytic site
• serve both for substrate recognition and for
initiating the next stage of the reaction, formation
of the transition state complex
C. The Transition State Complex
• For a reaction to occur, the substrates undergoing
the reaction need to be activated.
• If the energy levels of a substrate are plotted as the
substrate is progressively converted to product, the
curve will show a maximum energy level that is
higher than that of either the substrate or the
product
• The difference in energy between the substrate and
the transition state complex is called the activation
energy
• Enzymes increase the rate of the reaction by
decreasing this activation energy.
• They use various catalytic strategies, such as
electronic stabilization of the transition state
complex or acid-base catalysis, to obtain this
decrease
• Once the transition state complex is formed, it
can collapse back to substrates or decompose to
form products.
• The enzyme does not change the initial energy
level of the substrates or the final energy level
of the products.
Figure 5. Activation Energy Barrier
• Mechanisms of catalytic enzyme reaction
Mechanism of Actions of Enzymes
Mechanisms to facilitate catalysis : enzymes use
various combinations of general mechanisms to
achieve dramatic catalytic enhancement of the rates
of chemical reaction
• 1)Lowering of Activation Energy
• 2)catalysis by proximity
• 3)Acid-base catalysis
• 4)catalysis by strain
• 5) Covalent catalysis
• 6)Entropy effect
• 7)Product Substrate Orientation Theory
Mechanism of Actions of Enzymes cont’d
1) Catalysis by proximity: for molecules to react
,they must come within bond-forming distance
of one another. The higher their concentration,
the more frequent the encounter & the greater
the rate of reaction. when enzyme bind to the
active site ,it create a groove/pocket ,the
environment also orients the substrate molecule
spatially in a position ideal for them to
interact(rate enhancement)
Mechanism of Actions of Enzymes cont’d
• Emil Fisher model –proposed that enzymes and
their substrates interact to form an E-S (ES)
complex in lock and Key manner , hence thermal
stability> enzyme itself
• Daniel Koshland’s induced fit model: state that
when substrates approaches and bind to enzyme,
they induce a conformational changes, analoguos
to placing a hand(substrate) into a glove(enzyme)
Figure 6: Lock and Key model of enzyme-substrate bind
In this model, the enzyme changes shape on
substrate binding. The active site forms a shape
complementary to the substrate only after the
substrate has been bound
Figure 7: Induced-fit enzyme-substrate binding
Mechanism of Actions of Enzymes
cont’d
2) Lowering of Activation Energy:
• Enzyme lower the energy of activation
• Activation of energy is defined as the energy required
to convert all molecules of a reacting substance from
the ground state to the transition state
• Substrates are remaining in the energy trough, and are
placed at a higher energy level where upon
spontaneous degradation can occur
• During enzyme substrate binding ,weak interactions
between enzyme and substrate are optimized
Mechanism of Actions of Enzymes cont’d
• provides the major driving force for the
enzymatic catalysis
• Enzyme reduced the magnitude of activation
energy. This can be compared to making a
tunnel in a mountain, so that the barrier could
be lowered e.g activation energy for acid
hydrolysis of sucrose is 26,000cal/mol while
the activation energy is only 9,000cal/mol
when hydrolyzed by sucrase
Figure 8: Enzymes decrease the activation energy .Enzyme accelerate reactions
by decreasing free energy of activation ∆ G
Mechanism of Actions of Enzymes cont’d
3) Acid-Base catatysis:
• the ionization of functional groups of amino acyl side
chains and (where present) of prosthetic groups
contribute to catalysis by acting as acids or bases.
• Can either be specific or general
• Protonated form of histidine is an example of general
acid and its conjugate base, the general base.
• Histidine residues 12 and 119 at the active site of
ribonuclease function as acid and base in catalysis
• Histidine 12 acts as an acid and donates a proton to
form the basic form , then 2’-3’ cyclic phosphate is
formed
• Histidine 119 accepts a proton from the cyclic
Mechanism of Actions of Enzymes cont’d
• By specific-only protons H₃O⁺ or OH- ions, the rate
of reaction is sensitive to changes in the
concentration of proton but independent of the
concentrations of the acids(proton donor) or
base(proton acceptor) present in solution or at
active site
• HIV protease illustrates acid-base catalysis.
• In the enzymes of the aspartic protease family
include the digestive enzyme, pepsin, the lysosomal
cathepsins and the protease produced by HIV share
a common catalytic mechanism
• Here catalysis involves two aspartyl residues, which
act as acid-base catalysts
Mechanism of Actions of Enzymes cont’d
4) Substrate by Strain: binding of substrate to a preformed
site of the enzyme can induced strain in the substrate.
• The energy level of the substrate is raised
• A combination of substrate strain and acid-base catalysis is
seen in lysosome.
• The lysosome substrate has a repeating hexasaccharide
unit.
• Binding of substrate to the enzyme generates a strain
conformational change in the enzyme substrate complex
• In the transition state, acid catalyzed hydrolysis of the
glycosidic linkage by a glutamic acid residue at the active
site
• For example: carbonium ion on the N-acetyl neuraminic
acid residue, this result in change from transition state to
products
Mechanism of Actions of Enzymes cont’d
• The knowledge of transition state of an enzyme-
catalyzed reaction is frequently exploited by
chemists to design and create more effective
inhibitors called transition state analogs as
potential pharmacophores
5) Covalent catalysis: the process of covalent catalysis
involves the formation of a covalent bond between
enzyme and one or more substrates. The modified
enzyme then becomes a reactant
• E + S → E---S→ ES
• This mechanism introduces a new reaction pathway
whose activation energy is lower & therefore is
faster than the reaction pathway in homogenous
solution
Mechanism of Actions of Enzymes cont’d
• The chemical modification of enzyme is transient.
• On completion of the reaction, the return to its
original unmodified state common among
enzymes that catalyze group transfer reaction .
Residues on the enzymes that participate in
covalent catalysis generally are cysteine or serine
,occasionally histidine
• Chymotrypsin and fructose-2,6 bisphosphatase
illustrate covalent catalysis
Mechanism of Actions of Enzymes cont’d
6)Entropy effect: enzymes enhance reaction rates
by decreasing entropy. When correctly positioned
and bound on the enzyme surface , the substrate
are strained to the transition state-reffered to as
proximity effect
• Chemical reactions need physical apposition of
two reactants, collision between two substrate
molecules is determined by statistical probability.
• Since substrates ,usually are present in low
concentrations, the collision probability is less and
hence the reaction velocity is low
Mechanism of Actions of Enzymes
cont’d
7) Product substrate orientation Theory:
enzyme has appropriate three dimensional
structure to keep the substrate in a specific
orientation, such that the reactive group come
into physical apposition, leading to speedy
reactions; it has been shown that the hydroxyl
group of the 6th carbon atom of glucose and
the terminal phosphate group of ATP are
justaposed with the help of hexokinase
Factors Affecting Enzyme Actions
Factors affecting enzyme reaction
velocity
• The following factors influenced velocity of enzymatic
reaction :
a)Enzyme concentration
b)Substrate concentration
c)Hyperbolic shape of the Enzyme kinetic Curve
d)Temperature
e)pH- potential of hydrogen concentration
g)Modulator proteins :
i. Presence of activators,
ii. Presence of inhibitors
iii. Presence of repressor/derepressor
h)Covalent modification
Factors affecting enzyme reaction
velocity cont’d
• An organism must be able to control the catalytic
activities of its component enzymes, so that it can
coordinate its numerous metabolic processes,
respond to changes in its environment and growth
and differentiation, all in an orderly manner
a)Control of Enzyme Availability: the amount of a
given enzyme in a cell depend on both its rate of
synthesis and rate of degradation; which is directly
controlled by the cell. Various tissues of a higher
organism contain different sets of enzyme,
although most of its cells contain identical genetic
information
b)Control of enzyme activity: an enzymes
catalytic activity may be directly controlled
through conformational structural alternation
• The rate of an enzymatically catalyzed reaction
is directly proportional to the concentration of
its E-S complex ,which in turn varies with the
enzyme & [substrate] & with the enzyme
substrate-binding affinity
Figure 9. Effect of substrate concentration ( substrate saturation curve)
Figure 10. Effect of Enzyme concentration on Km
Figure 11. Effect pH on velocity of reaction
Figure 12. sigmoid -substrate-saturation kinetics of co-operative binding
Figure 13. Effect of temperature on velocity
Assignments
1). Describe the mechanism of catalytic actions of
enzymes
2). With the aid of illustrated diagram only,
describe the factors affecting enzyme activity
3). Write short note on the following:
a). Nature of enzymes
b). Enzymes with their coenzyme
c). metalloenzymes
Thank You
