Enzymology

Enzymology definition

• Branch of science that deals with study of

enzymes, their kinetics and function as well
as their relation to each other
 Enzymes
• Enzymes are biological catalysts that speed
up the rate of biochemical reactions
• Enzymes are produced by all living organisms
 Medical and biological importance of
enzymes
• Enzymes regulate physiological processes; defects in
enzyme function cause disease
• When cells are injured enzymes leak into plasma –
measurement of activity of such enzymes in plasma is an
integral part of modern day medical diagnosis
• Enzymes are used as drugs e.g pulmozyme used to
reduce mucous viscosity and enable the clearance of
airway secretions in patients with cystic fibrosis
• Enzymes are used as biosensors – e.g. glucose biosensors
use glucose oxidase or glucose dehydrogenase
 Medical and biological importance of
enzymes
• Immobilised enzymes which are enzymes
attached to solid supports are used in clinical
chemistry laboratories. E.g. glucose in urine is
measured used glucose oxidase
• Enzymes are used in the ELISA technique
Properties of/ General nature of enzymes

• Protein catalysts mediating all metabolic

reactions
-a catalyst does not change the chemical
reaction but it accelerates it
-they are not consumed in the overall reaction
but they undergo chemical or physical change
during the reaction
Properties of/ General nature of enzymes

• Enzymes differ from ordinary chemical catalysts

in that
(i) They have higher reaction rates. i.e. there are
more efficient compared to most catalysts
used in synthetic chemistry
(ii) Need milder reaction conditions
(iii) Have greater reaction specificity – capable of
relatively catalysing certain substrates whilst
discriminating against other molecules
Properties of/ General nature of enzymes

• Capacity for regulation

-catalytic activity of enzymes in response to
concentration of substances other than their
own substrates
-enzymes may be under some control e.g.
allosteric regulation, covalent modification,
transcriptional control
 Enzymes lower activation energy of
metabolic reactions
• The transitional state theory was proposed to
explain the action of a catalyst
• For a chemical reaction A B, energy is
required
• When energy is supplied A undergoes
transformation to a transitional state which is
an unstable state
• So it gets converted to product B which is more
stable
 Enzymes lower activation energy of
metabolic reactions
• The amount of energy that is
needed to convert a substance
from ground state to transitional
state is called activation energy.
• In the presence of a catalyst, A
transforms into a transitional
state very fast and requires less
energy
• Hence catalysts accelerates the
rate of reaction by decreasing
the energy of activation.
• Enzymes speed up reactions by
lowering energy of activation
 Enzyme specificity
Substrate specificity
• Enzymes are specific towards their substrates.
E.g. glucokinase catalyses the transfer of
phosphate to glucose. Galactokinase catalyses
transfer of phosphate group from ATP to
galactose.
• Though both enzymes catalyse the transfer of a
phosphate group from ATP they act on a
specific substrate
 Enzyme specificity
Reaction specificity
• A given enzyme catalyses only one specific
reaction. E.g. lipases only hydrolyse lipids,
urease hydrolyzes urea
• They do not catalyse an other type of reaction
Group specificity
• Some lytic (hydrolases) enzymes act on
specific groups
• Proteases are specific for peptide groups,
glycosidases are specific to glycosidic bonds
 Enzyme specificity
Absolute group specificity
• Certain lytic enzymes exhibit high order group
specificity.
• For example chymotrypsin is a protein
splitting enzyme
i.e. it hydrolyses peptide bonds in which
carboxyl group is contributed by aas
phenylalanine, tyrosine and tryptophan
 Enzyme specificity
Optical specificity
• Several enzymes exhibit optical specificity of
substrate on which they act
• It means they are able to recognise optical
isomers
 The active site
• That region of the enzyme molecule where the substrate binds and the catalytic
reaction occurs

• Usually a cleft or pocket on the surface of the enzyme , often on the interface of
two domains

• Usually only involves a small fraction of the enzyme surface

• Is complementary to the substrate shape and polarity

• Contains binding site for the substrate (attracts and positions substrate)

• Contains catalytic groups, these are the reactive side chains of aa’s or cofactors
which carry out the bonding/forming reactions involved

• Binding involves non covalent interactions (H-bonding, electrostatic

interactions, hydrophobic interactions, van der Waals interactions)

• Rest of the protein structure provides a superstucture to position the substrate

and catalytic groups, flexibilty for conformational changes, a means for
regulatory control and sites for recognition by other biomolecules
 The lock and key model
• Fit between the substrate and the active site of the
enzyme is exact
• Like a key fits into a lock very precisely
• The key is analogous to the substrate and the enzyme
analogous to the lock.
• A temporary structure called the enzyme-substrate
complex is formed
• Products have a different shape from the substrate
• Once formed, they are released from the active site
• Leaving it free to become attached to another substrate
Lock and key model

Enzyme Substrate Complex

 Induced fit model
• According to this model, the active site is flexible unlike rigid type
of the lock and key model.
• In the enzyme molecule the amino acid residues that make up
active site are not oriented properly in the absence of substrate.
• When substrate combines with enzyme, it induces conformational
change in the enzyme molecule in such a way that amino acids
that make up the active site are shifted into correct orientation to
favour tightly bound enzyme-substrate complex formation
followed by catalysis.
• The enzyme molecule is unstable in the induced conformation
and returns to its native conformation in the absence of
substrate.
Induced fit model
 Factors affecting enzyme activity
Temperature
• Like any chemical reaction, enzyme activity
increases with increase in temperature initially.
• After a critical temperature, the enzyme activity
decreases with increase in the temperature.
• When the effect of temperature on enzyme
activity is plotted, a cone-shaped curve is
obtained
• The figure indicates that there is an optimal
temperature at which enzyme is optimally active.
• It is known as the optimum temperature.
• For most of the enzymes, the optimum
temperature is the temperature of the cell or
body in which they occur.
 Factors affecting enzyme activity
Temperature cntd
• For example, human trypsin the
optimum temperature in 37 °C which
is the normal body temperature.
• The first half of the curve approaching
the optimum temperature indicates
that enzyme activity increased with
increase in the temperature due to
the increased kinetic energy of
reacting molecules
• The other half which corresponds to
decreased catalytic activity with
increased temperature is due to
denaturation of enzyme
 Factors affecting enzyme activity
pH
• Most of the enzymes are not maximally active throughout pH scale
(1-14).
• Several enzymes have optimum activity between pH of 5 to 9.
• When enzyme activity measured at several pH values is plotted a
bell shaped curve is obtained
• Since enzymes are proteins pH changes affects:
1. Charged state of catalytic site.
2. Conformation of enzyme molecules.
3. In addition low or high pH causes denaturation of enzymes. It
accounts for the less activity of enzymes at acidic or alkaline pH.
For most of the enzymes, optimum pH is the pH of body or cell in
which they occur. However, for some enzymes optimum pH may
not be in the neutral range
Factors affecting enzyme activity
pH
Optimum pH values

Enzyme
activity Trypsin

Pepsin

1 3 5 7 9 11
pH
 Factors affecting enzyme activity
Substrate concetration
• If the concentration of the substrate
(S) is increased while other conditions
are kept constant, the initial velocity
(Vo) (velocity measured when little
substrate is reacted) increases
proportionately in the beginning.
• As the substrate concentration
continues to increase, the increase in
Vo slows down and reaches maximum
Vmax and increases no further .
• The plot of (S) versus Vo is
rectangular hyperbola. It is called as
Michaelis plot
 Factors affecting enzyme activity
Modulators
• Inhibitors - are chemicals that reduce the rate of
enzymic reactions.
• They block the enzyme but they do not usually
destroy it.
• Enhancers- increase rate of the enzymatic
reaction
• The are usually specific and they work at low
concentrations.
 Nomenclature
•Enzymes commonly named with
(i)ase ending the name of the substrate e.g. urease catalyses hydrolysis of urea
(ii)
phrase describing the catalytic activity e.g.
DNA polymerase – polymerisation of nucleotides
Dercaboxylase – decarboxylase reactions
Protease – hydrolysis of proteins (peptides)
Non descriptive names also used e.g. catalase, pepsin. These enzymes were
named before the specific reaction catalysed was known
Catalase – dismutation of hydrogen peroxide
Pepsin – protease, endopeptidase
Trypsin – protease, endopeptidase
Lysozyme – lysis bacterial cell walls
 Enzyme Commission (E.C.) classification of
enzymes
• The E.C. numerical nomenclature classifies enzymes
based on the overall reaction catalysed
• It identifies classes of enzymes catalysing similar
reactions
• Besides giving very specific information about the
general reaction type, mechanism, substrates,
products and cofactors,
-the EC number prevents the confusion that would
otherwise result if only enzyme “common” names were
used in the literature.
 Enzyme Commission (E.C.) classification of
enzymes
• Since most enzymes have more than one (and often
many) common names,
E.g. catalase has the following names; caperase, catalase-
peroxidase, equilase, optidase.
-the use of EC numbers and/or EC-approved names allows
us to know exactly which enzyme is actually being referred
to
• Each enzyme is allocated a four-digit EC number, the first
three digits of which define the reaction catalysed and
the fourth of which is a unique identifier (serial number)
 What exactly does an EC number designate?

• The first digit of the EC classification code

denotes the general type of reaction
catalyzed by the enzyme and ranges from one
to six. This is the major class of the enzyme
 Reaction classes (Major Classes) in
the E.C. Nomenclature
Reaction Class Name Reaction catalyzed
1 Oxidoreductases Redox (oxidation/reduction)
reactions

2 Transferases Transfer of a functional group

from one molecule to
another
3 Hydrolases Hydrolysis: cleavage of a
bond by insertion of water
4 Lyases Bond elimination by means
other than hydrolysis or
oxidation

5 Isomerases Isomerization of molecules

(e.g., racemases and
epimerases)
6 Ligases Formation of new covalent
bonds at the expense of ATP
 What exactly does an EC number designate?

•  The second and third numbers are the

enzyme’s sub-class and sub-sub-class,
respectively, and describe the reaction with
respect to the compound, group, bond or
product involved in the reaction
• The fourth number is a unique identifier
(serial number)
 E.C. classification
• For example, histidine decarboxylase has the code EC
4.1.1.22, which breaks down as follows:
• 4, denotes a Lyase.
• 1, indicates it’s a carbon- carbon lyase C-C (Sub – class)
• 1, its a carboxy-lyase C-COOH.
• 22, arbitrary serial number.
 E.C. classification
Lyases (Major class)
Carbon-carbon lyases C-C (Sub class)
Carboxy-lyases C-COOH (Sub –sub class)
• The following ENZYME entries belong to class 4.1.1.-:
• 4.1.1.1 Pyruvate decarboxylase
• 4.1.1.2 Oxalate decarboxylase
• 4.1.1.3 Oxaloacetate decarboxylase
• 4.1.1.4 Acetoacetate decarboxylase
• 4.1.1.5 Acetolactate decarboxylase
 Systematic naming of enzymes
• An enzymes systematic name is used to prevent ambiguity.
• It is composed of the name of substrate(s) followed by a
word ending in “-ase” that specifies the type of reaction
catalyzed.
• For example, Malate dehydrogenase (EC 1.1.1.37)
interconverts L-malate and oxaloacetate using
nicotinamide adenine dinucleotide (NAD +) as a
coenzyme.  It’s systematic name “L-malate:
NAD+ oxidoreductase”, provides a brief chemical
description of the reaction it catalyzes
 Structure of enzymes
• All enzymes are globular proteins
• May be divided into two main groups
i) Simple enzymes – monocomponent
ii) Complex enzymes – bicomponent
Complex enzymes consist of a non protein part bound by either
covalent or non covalent forces

*Apoenzyme is inactive enzyme without its non protein component

*Holoenzyme is the optimal active enzyme with its non protein
component
 Cofactors
• Cofactors are non-protein molecules required
for activity of some enzymes
• Role of cofactors
-alter the three dimensional structure of the
protein
-alter the structure of the bound substrate to
activate the interaction with its enzyme
-actually participate in the chemical reaction by
acting as an acceptor or donor of a particular
group
 Cofactors cntd
• There are two types of cofactors:
- (i) Organic cofactors, e.g., flavin, pyridoxal
- (ii) Inorganic cofactors e.g., metal ions and
iron-sulfur clusters)
 Prosthetic groups
• Are cofactors that are tightly integrated into
an enzyme structure
• Prosthetic groups are distinguished by their
tight, stable incorporation into a protein’s
structure by covalent or non covalent forces
• Examples include pyridoxal phosphate,
thiamine pyrophosphate, biotin and the
metal ions Cu, Mg, Zn and Se
 Prosthetic groups cntd
• Metals are the most common prosthetic groups
• Enzymes that contain tightly bound metal ions are
termed metalloenzymes
• Metal ions that participate in redox reactions are
complexed to prosthetic groups such as heme
• Metals also facilitates the binding and orientation of
substrates, the formation of covalent bonds between
reaction intermediates or interaction to render them
electrophilic (electron poor) or nucleophilic (electron
rich)
Mechanisms of action of prosthetic groups

Transamination of pyridoxal dependant transaminase

• Amino transferases catalyse transamination reactions
• All aminotransferases have pyridoxal phosphate (PLP) as a
cofactor
• Pyridoxal phosphate (PLP) and its aminated form
pyridoxamine phosphate are tightly bound to
aminotransferases.
• Pyridoxal phosphate is bound to the enzyme both through
strong noncovalent interactions and through formation of a
Schiff base linkage involving a Lys residue at the active site
Mechanisms of action of prosthetic groups
Transamination of pyridoxal dependant transaminase
 Coenzymes
• Coenzymes are non protein organic molecules that
transport chemical groups from one enzyme to another
• They serve as substrate shuttles – or group transfer
reagents –that transport many substrates from their
point of generation to their point of utilisation
• Association with the coenzyme also stabilises substrates
such as hydrogen atoms/ hydride ions that are unstable
in the aqueous environment
• The hydride ion (H-) is carried by NAD or NADP+
• Other chemical groups transported by enzymes include
methyl groups (folates) and acylgroups (coenzyme Q)
 Coenzyme classification
i) Metabolite coenzymes synthesized from common
metabolites
-ATP can donate phosphate groups
-Adenosylmethionine donates methyl groups in many
biosythetic reactions
ii) Vitamin derived coenzymes – derivatives of vitamins.
-vitamins cannot be synthesised by mammals but must
be obtained as nutrients.
-deficiency of vitamin and corresponding coenzyme
deficit results in disease
Coenzymes
Mechanism of action of Coenzyme A
 Many coenzymes, prosthetic groups are
derivatives of vitamins
• Niacin (Vit B3) – precursor of NAD and NADP
involved in redox reactions
• Riboflavin (Vit B2) – ingested and converted
to flavin mononucleotides (FMN) and flavin
adenine dinucleotide (FAD)
• Pyridoxal phosphate (Vit B6) – precursor of
pyridoxal phosphate involved in
transamination reactions