Welcome to class of

Basic enzymology

Dr. Meera Kaur

 Learning Objectives
• To understand
– Enzyme and how they differ from nonbiological catalyst

– Properties of enzymes and principles underlying their catalytic

power

– The classifications of enzymes

– The factors determining the rate of a reaction (activation

energy)

– Enzyme’s role in lowering the activation energy in a reaction

– The lock and key model to explain the binding and specificity
in enzyme action
 What is an Enzyme?
• Enzymes
- Are the most remarkable and highly specialized protein

- Catalyze hundreds of stepwise reactions in biological systems

- Regulate many different metabolic activities necessary to sustain life

In short,
Living systems use catalyst called enzymes to increase the
rate of chemical reactions
 Why to study enzymology
The study of enzymes has immense practical importance:
- In medical science: to know the epidemiology, to diagnose, and to
treat diseases ( inheritable genetic disorders)

- In chemical industries

- In food Processing

- In agriculture

- In everyday activities in the home ( food preparation, cleaning,

beauty care etc.)
 A note on nomenclature…
• The International Enzyme Commission (EC) has recommended
a systematic nomenclature for enzymes.

• This commission assigns names and numbers to enzymes

according to the reaction they catalyze. An example of
systematic enzyme name is EC 3.5.1.5 urea aminohydrolases
for the enzyme that catalyzes the hydrolysis of urea.

• The name of an enzyme frequently provides a clue to its

function. In some cases, an enzyme is named by incorporating
the suffix -ase into the name of its substrate, e.g., pyruvate
decarboxylases catalyzes the removal of a CO2 group from
pyruvate
 A note on nomenclature
• Certain protein cutting digestive enzymes are exception to this
general rule of enzyme nomenclature, e.g., pepsin, trypsin,
chymotrypsin and thrombin.

Isozyme: Within an organism more than one enzyme may

catalyze a given reaction. Multiple enzymes catalyzing the same
reaction is called isozymes.

- isozymes differ in their catalytic properties. Consequently,

the various isozymes that are present in different tissues or at
different developmental stages can carry out slightly different
metabolic functions.
Pyruvate decarboxylase
Alanine aminotransferase
 Enzyme classification
Class of Enzyme Type of reaction catalyzed

1. Oxidoreductases Oxidation-reduction reactions

2. Transferases Transfer of functional groups

3. Hydrolases Hydrolysis reactions

4. Lyases Group elimination to form double bond

5. Isomerases Isomerization reactions

6. Ligases Bond formation couples with ATP hydrolysis

 Enzymes vs. Nonbiological catalysts

• Enzymes have their reaction specificity

• The activities of many enzymes are regulated so that

the organism can respond to the changing condition
or follow genetically determined developmental
programs

• Enzyme activity is affected by the temperature and

pH of the medium
 Properties of enzymes…
• All enzymes are proteins or protein in nature (except a small group
of catalytic molecules)

• Enzymes have molecular weight ranging form 20,000 - 1 million

• Some enzymes require no chemical group other than their amino

acid residue for activity.

• Some enzymes may require an additional chemical compound

called cofactors (Zn2+, Mg2+, Fe2+, Mn2+etc.)

• Some enzymes may require a complex orgainc or metelloorganic

molecule called coenzymes

• Sometimes they need both cofactors and coenzymes

 Properties of enzymes
• A coenzyme or metal ion that is covalently bound to the enzyme
protein is called a prosthetic group

• A complete catalytically active enzymes together with its

coenzymes and or metal ions is called a holoenzyme

• The protein part of such an enzyme is called the apoenzyme or

apoprotein

• Enzymes are classified by the reactions they catalyze. Many

enzymes have been named by adding the suffix “-ase” to the
name of their substrate or to a word or phrase describing their
activities. Thus urease catalyzes hydrolysis of urea.
 How do enzymes work ?
• An enzyme circumvents many problems providing a
specific environment within which a given reaction is
energetically more favorable.

• An enzyme-catalyzed reaction occurs within the

confinement of a pocket on the enzymes called the
active site

• The molecule that is bound by the active site and

acted upon by the enzyme is called the substrate

• The enzyme-substrate complex is central to the

action of enzymes.
 Enzyme specificity
Enzymes are specific and efficient catalyst

In nonenzymatic reactions, substrates are transformed into product

by bond-making and bond-breaking processes. In an enzymatic
reaction, these processes occur through interactions of the
substrates with appropriate side chains of the amino acid
residues of the enzyme or with the coenzyme of the enzyme.
- One property of many enzymes is specificity: catalyzing
one chemical reaction with only one substrate

An Example is Carbonic anhydrase. This enzyme catalyzes only

one reversible reaction, the breakdown of carbonic acid (H2CO3)
to water and carbon dioxide. At ideal conditions, a single
molecule of Carbonic anhydrase is capable of catalyzing the
breakdown of about 36 million molecules of carbonic acid in one
minute.
 How do enzymes work?
• In a biological reaction, the reacting species must come together
and undergo electronic rearrangement that result in the
formation of a product. Let us consider an idealized transfer
reaction in which compound A—B reacts with compound C to
form two new products, A and B—C.

• In order for the first two compounds to react, they must

approach closely enough for their constituent atoms to interact.

• Normally, atoms that approach too closely repel each other; but
if the groups have sufficient free energy, they can pass this point
and react with each other to form products.

• The energy requiring step of the reaction is known as an energy

barrier, called as free energy of activation or activation energy
(G‡)
Idealized transfer reaction
Reaction coordinate diagram for the reaction A—B + C A + B—C

The progress of the

reaction(reaction
coordinate) is shown on
the horizontal axis, and
the free energy(G) is
shown on the vertical
axis. The transition
state of the reaction,
represented as a..b..c, is
the point of highest free
energy.The free energy
difference between the
reactant and the
transition state is the free
energy of activation
(G‡. ).
 Reaction coordinate diagram of a reaction:
Here reactants and products have different free energies
When the free energy of the reactant is greater than that of the product, the free
energy changes for the reaction is negative, so the reaction proceeds
spontaneously (a). When the free energy of the product is greater than that of the
reactant, the free energy change of the reaction is positive, so the reaction does
not proceed spontaneously (however the reverse reaction does proceed).
Effect of a catalyst on a chemical reaction

Here the reactants

proceed through a
transition state (X‡)
during their conversion
to products. A catalyst
lowers the free energy
of activation ( G‡) for
the reaction so that 
G‡cat < G‡uncat. Loweing
the free energy of the
transition state (X‡)
accelerates the reaction
because more reactants
are able to achieve the
transition state per unit
time
 Enzymes use chemical catalytic mechanism

• There are three basic kinds of chemical catalytic

mechanisms used by enzymes:

- Acid-base catalysis

- Covalent catalysis

- Metal ion catalysis

 Acid-base catalysis

• In acid-base catalysis a proton is transferred between

the enzyme and the substrate

• It may be either acid catalysis or base catalysis or both

• Many enzymes use both

 Tautomerization of a ketone

Tautomers are interconvertible isomers that differ in the placement of a

hydrogen and a double bond. The dotted lines represent bonds in the process
of breaking or forming.
 Acid catalysis

If a catalyst (H—A) donates a proton to the to the keton’s oxygen

atom, it reduces the unfavorable character of the transition state,
thereby lowers its energy. Hence, it also lowers the activation energy
barriers for the reaction. This is an example of acid catalyst, since the
catalyst acts as an acid by donating a proton.
 Keto-enol tautomerization (base catalysis)

The same keto-enol tautomerization reaction shown in the previous slide can be
accelerated by a catalyst that can accept a proton. In this case, it is a base
catalyst (:B), where the dots represent unpaired electron. Base catalysis lowers
the energy of transition state and thereby accelerates the reaction.
Amino acid side chains that can act as acid-base catalysts
 Covalent catalysis…
• In covalent catalysis, a covalent bond is formed between the catalyst
and the substrate during formation of the transition state

• In covalent catalysis, an electron rich group in the enzyme active site

forms a covalent adduct with a substrate. This covalent complex is
much more stable than a transition state and can be isolated.

• Covalent catalysis is also called nucleophilic catalysis, because the

catalyst is a nucleophile (an electron-rich group in search of a electron
-poor center ( a compound with an electron deficiency is known as an
electrophile)

• Enzymes that use covalent catalysis undergo a two-part reaction

process. So the reaction coordinate diagram contains two energy
barriers with the reaction intermediate between them.
Protein groups that can act as covalent catalysts
 Reaction coordinate diagram for a
reaction accelerated by covalent catalysis

Two transition
states flank the
covalent
intermediate.
The relative
heights of the
activation energy
barriers (the
energies of the
two transition
states, X‡1 and
X‡2) vary
depending on
the reactions.
 Metal ion catalysis…

• Metal ions participate in enzymatic reactions by

mediating oxidation-reduction reactions, or by
promoting the reactivity of other groups in the
enzyme’s active site through electrostatic effects

• A protein bound metal ion can interact directly with

the reacting substrate
 Metal ion catalysis

Example: The conversion of acetaldehyde to ethanol catalyzed by the liver

enzyme alcohol dehydrogenase. Here a zinc ion stabilizes the developing
negative charge on the oxygen atom during formation of the transition state.
 The unique properties of enzyme catalyst

• Enzyme–substrate complexes:

– Enzymes are specific and efficient catalyst

– Lock and key fit of enzymes and substrates help produce

enzyme specificity
Lock-and-key model of enzyme action

In this early model, the substrate

(key) was believed to bind tightly to
a site on the enzyme (lock) that was
exactly complementary to it in its
size, shape and charge.
 Enzyme-substrate complexes
Lock and key fit of enzymes and substrates helps produce enzyme
specificity: A substrate molecule must contact an enzyme molecule before it
can be transformed into products. Once the substrate has made contact, it must
bind to the enzyme at the active site— a place where the conversion of
substrate to product can take effect. The active site is usually a dimple, pocket
or crevice formed by fold in the chain conformation of the protein. When the
substrate key fits the enzyme lock, an enzyme-substrate complex is formed.

The fit between enzyme and substrate in binding helps to produce enzyme
specificity. Complementarity between enzyme and and substrate is governed by
factors besides shape– e.g., the electrical charge. The substrate may be
positively charged and the enzyme’s active site negatively charged and vice
versa.

Hydrogen bonds and other weak forces may also hold the the enzyme- substrate
complex together. Hydrophobic regions of the substrate associate with similar
regions on the active site of the enzyme. For most enzymes contributions from
shape, charge, and a large number of weak forces are responsible for the
formation and strength of the complex.
 Transition state stabilization
• Linus Pauling (1946) formulated the principle that

– An enzyme increases the reaction rate not by binding tightly

to the substrates, but by binding tightly to the reaction’s
transition state, i.e., substrate that have been strained
toward the structure of the product. In other word, the tightly
bound key of the lock and key model is the ‘transition state’,
not the substrate.

Pauling’s theory of enzyme’s actions is consistent with the

chemical catalytic mechanisms, in which the catalyst
stabilizes the transition state of the reaction. In an enzyme,
tight binding( stabilization) of the transition state occurs in
addition to acid-base, covalent, or metal ion catalysis.
Effect of very tight substrate binding on enzyme catalysis
The red line indicates the free
energy changes as the
reactants pass through a
transition state to products.
The blue line indicates free
energy changes for the same
reaction when the reactants
(substrates) are very tightly
bound to the enzyme ( they
are at a very low free energy
level) before they react. The
activation energy barrier (G‡)
is therefore increased
because the tight binding
must be overcome before the
substrates can reach the
transition state.