ENZYMOLOGY

Almost every significant life process is dependent on enzymes activity. Enzymes are biological
catalysts responsible for supporting almost all of the chemical reactions that maintain animal
homeostasis.

Enzymes are found in all tissues and fluids in the body. Intracellular enzymes catalyse the reactions
of metabolic pathways. Plasma membrane enzymes regulate catalysis with in cells in response to
extrance molar signals, and enzymes of the circulatory system are responsible for regulating the
clotting of blood.

Like all catalysts, enzymes take part in the reaction - that is how they provide an alternative
reaction pathway. But they do not undergo permanent changes and so remain unchanged at the
end of the reaction. They can only alter the rate of reaction, not the position of the equilibrium.

Most chemical catalysts catalyse a wide range of reactions. They are not usually very selective.
In contrast enzymes are usually highly selective, catalysing specific reactions only. This
specificity is due to the shapes of the enzyme molecules.

All enzymes are protein in nature except for a class of RNA modifying catalysts known as
ribozymes (molecules of RNA that catalyse reactions on the phosphodiester bond of other RNAs).

Enzyme classification
• Initially, enzymes were given trivial nomenclature.
Some enzymes retain their early trivial names which give no hint of the associated
enzymatic reaction e.g. trypsin, pepsin, chymotrypsin.
• Other have been named by adding the surfix – ase to the name of the substrate e.g. urease
for urea hydrolysis, Ribonuclease for breakdown of ribonucleic acid ( RNA).
• Most enzymes catalyze the transfer of electrons, atoms or functional groups.
In the old system of classification, they were given code number and assigned names e.g.
hexokinase
ATP + D- glucose → ADP + D- glucose -6-phosphate
( glucokinase)
The former systematic name is ATP: D- glucosephasphotransferase meaning that it catalyses the
transfer of a phosphate group from ATP to glucose its classified as 2.7.1.1.
1st digit - class - 2 - transferase
2nd digit - sub class - 7- phosphotransferases
rd
3 digit - sub sub class - 1- phosphotranferases with OH
4th digit - serial number -1- glucose as the phosphate group acceptor
• Because of the above, its happened that the same enzyme is known by 2 or more names or
that 2 different enzymes have been given one name. Due to these and other ambiguities as
well as the ever increasing newly discovered enzymes, a systematic basis of naming and
classifying enzymes has been adopted by the International Union of Biochemists basing on
the reactions they catalyze.
A systematic classification and nomenclature for enzyme catalyzed reactions has then been
established by the commission of enzymes of the Interactional Union of Biochemistry (IUB). Here
enzymes are placed in 6 groups.
No. Class Reaction catalysed
1. Oxidoreductases Transfer of electrons
2. Transferases Group transfer of electrons
AX + B → A + Bx
3. Hydrolases Hydrolytic reactions
( transfer of functional groups to water)
AX + H2O → AH + X-OH
4 Lyases Addition of groups of double bonds or vice versa ( formation or
elimination of double bonds)
5. Isomerases Isomer formation i.e. transfer of groups within a molecule.
6. Ligases Formation of C-C, C-S, C-0 and C-N bonds by condensation reactios
compled with ATP cleavage on a similar triphasphate.

Properties of Enzymes.
i. They are catalysts. They increase the velocity of chemical reactions and aren’t
consumed during the reactions they catalyze. They help to overcome the barrier
between reactants and products which prevents the spontaneous occurrence of these
reactions and allowing the reactions to occur at rate appropriate to the needs of the cell.
ii. They are exceedingly efficient under optimal conditions. They have a high turnover.
Turnover is the number of substrate molecules metabolized per enzyme molecule per
minute. For most enzymes, its approximately 1000 and in extremes may be greater than
1M.
iii. Most enzymatic reactions are specific in terms of the nature of the reaction catalyzed
and the structure of the substrate catalyzed.
iv. The spectrum of the reactions catalyzed is broad. Enzymes are responsible for
catalyzing polymerization reactions, redox reactions, dehydration reactions,
condensation reactions, acyl transfer reactions and free radical reactions.
v. Enzymes themselves are a subject to a variety of cellular controls. Enzyme activity can
be inhibited i.e activated or inactivated by binding allosteric modifiers ( effectors) or
by covalent modification of the enzyme itself.
vi. They may be present in both active and inactive forms (zymogen) and the rate and
extent of the interconversion is influenced by the composition of the environment at
any instant.
vii. They are protein in nature. The proteins in enzymes are usually globular and hence are
pH and temp sensitive as these affect their chemical and molecular nature.
viii. for activity, some enzymes require an additional chemical component known as a
cofactor. The co-factor may be
• organic groups that are permanently bound to the enzyme (prosthetic groups)
• cations - positively charged metal ions (activators like Fe2+, Mg2+, Zn2+), which
temporarily bind to the active site of the enzyme, giving an intense positive
charge to the enzyme's protein
 • organic molecules, usually vitamins or made from vitamins (coenzymes),
which are not permanently bound to the enzyme molecule, but combine with
the enzyme-substrate complex temporarily.
ix. A catalytcally active enzyme with its co-factor (metal ion or co-enzyme) is called a
Holo enzyme. An enzyme without a cofactor is called an Apo enzyme. Co-enzymes
and metal ions are stable to heat but the Apo enzyme can be denatured by heat. Apo
Enzymes are also inactive in the absence of a cofactor.

How enzymes work

For two molecules to react they must collide with one another. They must collide in the right
direction (orientation) and with sufficient energy. Sufficient energy means that between them
they have enough energy to overcome the energy barrier to reaction. This is called the activation
energy.
Enzymes have an active site. This is part of the molecule that has just the right shape and
functional groups to bind to one of the reacting molecules. The reacting molecule that binds to
the enzyme is called the substrate.
An enzyme-catalysed reaction takes a different 'route'. The enzyme and substrate form a reaction
intermediate. Its formation has a lower activation energy than the reaction between reactants
without a catalyst.
A simplified picture
Route A reactant 1 + reactant 2 product

Route B reactant 1 + enzyme intermediate

intermediate + reactant 2 product + enzyme
In Route B the enzyme is used to form a reaction intermediate which is at lower energy level
compared to Route A, but when this reacts with another reactant the enzyme reforms and the
same product is formed, but at a lower energy level compared to Route A.

Lock and key hypothesis

This is the simplest model to represent how an enzyme works. The substrate simply fits into the
active site to form a reaction intermediate.

Induced fit hypothesis

In this model the enzyme molecule changes shape as the substrate molecules gets close. The
change in shape is 'induced' by the approaching substrate molecule. This more sophisticated
model relies on the fact that molecules are flexible because single covalent bonds are free to
rotate.

Factors affecting catalytic activity of enzymes

1. Temperature

As the temperature rises, reacting molecules have more and more kinetic energy. This increases
the chances of a successful collision and so the rate increases. There is a certain temperature at
which an enzyme's catalytic activity is at its greatest (see graph). This optimal temperature is
usually around human body temperature (36.5 oC) for the enzymes in human cells.
Above this temperature the enzyme structure begins to break down (denature) since at higher
temperatures intra- and intermolecular bonds are broken as the enzyme molecules gain even
more kinetic energy.
2. pH

Each enzyme works within quite a small pH range. There is a pH at which its activity is greatest
(the optimal pH). This is because changes in pH can make and break intra- and intermolecular
bonds, changing the shape of the enzyme and, therefore, its effectiveness.
3. Concentration of enzyme and substrate
The rate of an enzyme-catalysed reaction depends on the concentrations of enzyme and substrate.
As the concentration of either is increased the rate of reaction increases (see graphs).
For a given enzyme concentration, the rate of reaction increases with increasing substrate
concentration up to a point, above which any further increase in substrate concentration produces
no significant change in reaction rate. This is because the active sites of the enzyme molecules at
any given moment are virtually saturated with substrate. The enzyme/substrate complex has to
dissociate before the active sites are free to accommodate more substrate. (See graph)
Provided that the substrate concentration is high and that temperature and pH are kept constant,
the rate of reaction is proportional to the enzyme concentration. (See graph)

Active site
The active site of an enzyme is that region on an enzyme molecule that binds substrate and contains
the residues that directly participate in the making and breaking of bonds. These residues are called
catalytic groups. The active site takes up a relatively small part of the total volume of an enzyme
molecule.
Substrates are bound to enzymes by multiple weak attractions. Reversible interactions of
biomolecules and enzyme molecules are mediated by H-bonds, electrostatic bonds, van-der-waals
forces and hydrophobic interaction.
Active sites are crevices which because of the chemical groups in them create a microenvironment
in certain of these residues acquire special properties for catalysis.
The specificity of binding depends on this precisely defined arrangement of atoms in the active
site i.e the shape of the active site is complimentary to that of the substrate and this is explained as
lock and key.
But sometimes convenient ability of the active site and substrate come about after the substrate is
bound to the active site. This dynamic recognition process is explained as or by induced – fit
hypothesis. This strengthens the binding and there are 4 possible mechanisms of catalysis.

Mechanism of catalysis
1. Catalysis by bond strain. The induced structural rearrangements produce strained substrate
bonds that attain transition state more easily.
2. Covalent catalysis. A covalent intermediate develops between substrate and enzyme.
3. Catalysis involving acids and bases. The use of acidic and basic groups, to form interactions
at the active site.
4. Catalysis by orientation and proximity. Enzyme substrate interactions induce reactive groups
into proximity with one another.

Enzyme regulation.
1. Negative feedback. The enzyme that catalyses the first step in a biosynthetic pathway is usually
inhibited by the ultimate product. This is called feedback inhibition.
A → B → C → K ( End product).
Enzyme that converts A to B is inhibited by K.
When the concentration of K reaches a sufficiently high level the rxn stops.
2. Positive feed forward. The presence of a substrate activates the enzyme to covert the substrate
to a product. e.g. substrate A activates the enzyme to convert it to product B.
3. Proteolytic cleavage. Some enzymes are produced in an inactive form called a Zymogen, or
precursors which can be activated at a physiologically appropriate time and place e.g. trypsinogen
produced from the pancreas is activated by peptide bond clearage in the small intestines to form
trypsin which is active, this is termed proteolytic activation.
4. Suicide inhibition. This is where an enzyme converts a substrate into a reactive inhibitor that
immediately inactivates its catalytic activity e.g. fluorouracil which is used in cancer treatment i.e.
its an anti –cancer.
5. Regulation of gene expression. This controls the quantity and rate of enzyme synthesis.
Enzymes exist in a small concentration, but this concentration increases in the presence of the
substrate through expression of the enzyme’s gene.
6. Half life / rate of degradation. Enzymes have different half lives and are supposed to work
within a given period of time and then be degraded, so as to end the metabolic reactions which
they catalyse.
7. Covalent modification. Some enzymes are activated by covalently adding a particular bond, or
covalently removing a bond. e.g. phosphorylation and dephosphorylation of enzymes by glucagon
and insulin hormones respectively.

Enzyme Kinetics
Michaelis-menten equation
It helps us to establish the affinity of an enzyme for a substrate
It also helps to determine the intracellular substrate concentration
It also helps to identify the enzyme, when Km is known
V = Vmax [S]
Km + [S]
Where v =velocity of reaction
Vmax = maximum rate achieved by the system
[S] = concentration of the substrate
Km = Michaelis constant
Through enzyme kinetics, this equation is the rate equation for a one-substrate enzyme catalysed
reaction. It relates the initial reaction rate, the maximum reaction rate and the initial substrate
concentration, through the Michaelis constant ( a measure of the substrate-binding affinity.)
Since Vmax α affinity i.e affinity α 1
Km Km
Thus affinity is inversely proportional to Km. The lower the Km, the higher the affinity of the
enzyme for the substrate and vice versa.
Inhibition of enzyme activity
Some substances reduce or even stop the catalytic activity of enzymes in biochemical reactions.
They block or distort the active site. These chemicals are called inhibitors, because they inhibit
reaction.
Enzyme inhibition reduces the rate of enzyme catalysed reactions. There are 2 types of inhibitions.
1. reversible inhibitors: These are be removed from enzyme by dialysis
2. Irreversible inhibitors: Can’t be removed by dialysis. They permanently cling on the
enzyme.
There are 3 types of reversible inhibitors.
1. Competitive Inhibitors. These are Inhibitors that occupy the active site and prevent a
substrate molecule from binding to the enzyme and are said to be active site-directed (as they
'compete' with the substrate for the active site).
This inhibition can be overcome by increasing the concentration of the substrate. The effect of the
competitive inhibitor depends on the concentration of the inhibitor, substrate, and the relative
affinity for the enzyme of the 2.
The presence of the inhibitor has no effect on Vmax but it increases Km because as substrate
concentration increases even the enzyme molecules are in contact with the competitive inhibitor
will finally release it and form contact with the substrate. At a sufficiently high [S], inhibition
(competitive) is reversed because all the enzyme molecules are busy with the substrate molecules
and V max is attainable.
Lineweaver-Burke plot for competitive Inhibition

2. Non Competitive Inhibitors. These are Inhibitors that attach to other parts of the enzyme
molecule (such as the allosteric site), perhaps distorting its shape and are said to be non-active
site-directed.
As shown by the lineweaver-Burke plot, these inhibitors decrease Vmax, where as Km
remains unchanged.
Lineweaver-Burke plot for non competitive Inhibition,
3. Uncompetitive Inhibitors. Also known as anti-competitive inhibitors, bind only to the
complex formed between the enzyme and the substrate. These occur in reactions with 2 or more
substrates or products.
These decrease Vmax and increase Km.
Lineweaver-Burke plot for uncompetitive

Clinical significance
Enzymes are essential to diagnosis and treatment of almost all diseases.
- Damage of tissues leads to release of intracellular enzymes into the general circulation.
- In cardiac infarction, liver cirrhosis  other diseases, circulating plasma concentration of
enzymes specific to these tissues is measured.
- Combat of disease e.g. salicylates and their consertives (aspirin) inhibit cyclooxygenase (
which initiates the conversion of prachidonic acid to thromboxanes  prostaglandins)
secondary messangers in Rheamatoid arthritis).
- Restriction endonucleases are used in analysis of the nucleotide sequences of genes, where
they hydrolyse DNA at specific nucleotide sequences known as restriction sites. This is
the basis of cataloging many genetic diseases.
- Enzyme therapy