Enzyme catalysed reactions Enzyme kinetics, Enzyme's mechanism of action.

1. Enzyme
Enzymes are bio-catalyst, they are catalysts of life. Enzyme may be defined as a catalyst
that regulates the rate at which chemical reactions proceed in living organisms without
itself being altered in the process. They are protein in nature, (exception; RNA acting as
ribosome), colloids and thermolabile in character and specific in their action.
Example: Glycosides, Sucrose, Pepsin etc.
Classification of enzymes:
The classification of enzymes are given below: -
i. Oxidoreductase: e.g.- Alcoholdehydrogenase, Cytochrome-Oxidase, Land O-
amino acid Oxidase.
ii. Transferase: e.g.- Hexokinase, Transaminases, Phosphoryl trans-methylases.
iii. Hydrolase: e.g.- Lipase, Choline-esterase, Acid and alkaline- Phosphatares, Pepsin
ureas.
iv. Lyase: e.g.- Aldolase, Fumarase, Histidase.
v. Isomerase: e.g.- Triose Phosphate isomerase, Retional isomerase, Phosphotiexose
isomerase,
vi. Ligases: e.g.- Acetyl CoA.
Enzyme catalyzed reactions:
i) Oxidoreductases: are involved in redox reactions.
Eg. - Dehydrogenase (removal of hydrogen)
- Oxidase (add oxygen to hydrogen, forming water)

2. ii) Transferases: They transfer a group of
atoms form one molecule to another
General equation:
A-X + B ↔ BX + A
transaminase (transfer amino group from one
molecule to another)
phosphotransferase (transfer of phosphate group)
iii) Hydrolases: catalyze hydrolysis of
substrate by addition of water.
General equation:
A-X + H2O ↔ X-OH + HA
Eg.
- maltase (maltose broken to 2 glucose)
- lipase (lipid broken to fatty acid and glycerol)
iv) Lyases: breaks chemical bonds without adding water.
General equation:
A-B → A=B + X-Y
Eg.
- Decarboxylases (remove carboxyl group from
respiratory substrates to release carbon dioxide)
v) Isomerases: catalyzes conversion of one isomer to
another by transferring a group of atoms from one
molecule to another.
Eg.
- Triose phosphate isomerase.
vi) Ligases: catalyzes the synthesis of new chemical bonds, using ATP.
Eg.,
- DNA ligase is involved in DNA synthesis

3. Enzyme Kinetics:
Reaction Model: The enzyme reacts with the substrate by binding to its active site to
form the enzyme substrate complex,ES. Thatreaction followed by thedecomposition of
ESto regeneratethefreeEand the new product P.
Michaelis-Menten Equation:
Here, Vo =Initialreactionvelocity
Vmax = Maximumvelocity
Km = Michaelis constant =
K−1 + K2
K1
Assumption 1: (Rate formation of ES complex)
Rate formation,
Assumption 2: (Rate of breakdown ES complex)
-
{“- “negative sign indicates reduction of conc. Of [ES] complex with time}
Assumption 3: (Steady state assumption)
The rate of breakdown of ES complex very rapidly equal to the rate of formation ES
Thus,
Or, K1 [E] [S] = K– 1 [ES] + K2 [ES] {Fromtheequation1andequation}
Or, K1 [E] [S] = (K1 + K2) [ES]
Steadystateassumption→ [ES]isconstant.
So, Formation of ES = Loss of ES.

4. We know that–
Michaelis constant Km is the substrate concentration at which the reaction rate is at half
maximum & is an inverse measure of the substrate’s affinity to the enzyme.
So,
Thetotalamountofenzymeinthesystemmustbethesamethroughouttheexperimentbut
it can either be free (unbound) E or in complex with substrate, ES. If we term the toral
enzyme Et, this relationship can be written out:
[Et] = [E]+ [ES]----------(4)
From equation 3 and equation 4 we can write-
Or,[Et][S]–[ES][S]=KM [ES]
Or,[Et][S]=KM [ES]+[ES][S]
Or, [Et] [S] = ([S] + KM) [ES]
Vo is determined by the breakdown of ES to form product, which is denoted by [ES].
Vo = K2 [ES]--------------(6)
From the equation 5 and equation 6 we can write,
The maximum rate, which can call Vmax, would be achieved when all of the enzyme
molecules havesubstratebound.Undertheconditionswhen[S]ismuchgreaterthan[E],
itisfairtoassume that all E will be in the form ES. Therefore [Et]= [ES]. Thinking again
about equation 6, we could substitute the term Vmax for Vo and [Et] for [ES]. This
would give us –
Vmax = K2 [Et]

5. Fromequation7,
Mechanism of action:
The catalytic efficiency of enzyme is explained by 2 perspectives:
1. Thermodynamic changes,
2. Processes at the active site.
Thermodynamic changes:
Substrate Products
- Acquire a transitional state
- The difference in energy level of transitional state and substrate is called activational
barrier:
- Only few substrates can cross this barrier
to be converted to product.
- That is why rate of uncatalyzed reaction
is much slow.
- When enzyme present it provides an
alternative pathway for conversion of
substrate into product.
- Enzyme accelerate reaction rate by
providing transition state with low activational energy for formation of product.
- Hence, reaction rate is enhanced by many folds in the presence of enzymes.
- The total energy of the system remains the same and equilibrium state is not
disturbed.
Processes at the active site:
The types of catalytic mechanisms that enzymes employ have been classified as:
1. Acid–base catalysis 3. Metal ion catalysis
2. Covalent catalysis 4. Proximity and orientation effects
5. Catalysis by approximation.

6. 1. Acid-base catalysis:
➢ The catalytic activity of these enzymes is sensitive to pH, since the pH influences the
state of protonation of side chains at the active site.
➢ RNase A Is an Acid–Base Catalyst. Bovine pancreatic RNase A provides an example
of enzymatically mediated acid–base catalysis.
➢ This digestive enzyme is secreted by the
pancreas into the small intestine, where it
hydrolyzes RNA to its component nucleotides.
➢ RNase A has two essential His residues, His 12
and His 119, that act in a concerted manner as
general acid and base catalysts. Evidently,
RNase A
2. Covalent catalysis:
❖ The process of covalent catalysis involves the formation of a covalent bond
between the enzyme and one or more substrates.
❖ Covalent catalysis accelerates reaction rates through the transient formation of a
catalyst-substrate covalent bond.
❖ Usually, this covalent bond is formed by the reaction of a nucleophilic group on the
catalyst with an electrophilic group on the substrate, and hence this form of
catalysis is often also called nucleophilic catalysis.
❖ Examples of enzymes that participate in covalent catalysis include the proteolytic
enzyme chymotrypsin and trypsin in which the nucleophile is the hydroxyl group
on the serine.

7. 3. Metal ion catalysis:
❖ Nearly one-third of all known enzymes require metal ions for catalytic activity.
This group of enzymes includes the metalloenzymes.
❖ Most common transition metal ions include Fe2+
, Fe3+
,
Cu2+
, Mn2+
or, Co2+.
❖ Ionic interactions between an enzyme-bound metal and a
substrate can help orient the substrate for reaction or
stabilize
❖ An excellent example of this phenomenon occurs in the
catalytic mechanism of carbonic anhydrase a widely
occurring enzyme that catalyzes the reaction:
CO2 + H2O ⇌ HCO− 3
+ H+
4. Proximity and orientation effects:
❖ Enzyme-substrate interactions align the reactive chemical groups and hold them
close together in an optimal geometry, which increases the rate of the reaction.
❖ This reduces the entropy of the reactants and thus makes addition or transfer
reactions less unfavorable, since a reduction in the overall entropy when two
reactants become a single product.
5. Catalysis by approximation:
❖ Many reactions include two distinct substrates. In such cases, the reaction rate may
be considerably enhanced by bringing the two substrates together along a single
binding surface on an enzyme.
❖ NMP kinases bring two nucleotides together to facilitate the transfer of a
phosphoryl group from one nucleotide to the other.
❖ This strategy takes advantage of binding
energy and positions the substrates in the
correct orientation for the reaction to
proceed.
❖ An example of catalysis by approximation is
when NMP kinases bring two nucleotides
together to facilitate the transfer of a
phosphoryl group from one nucleotide to the
other.