BCH 201 – GENERAL BIOCHEMISTRY 1 – (3 UNITS)

DR AKINLOYE’S ASPECT

ENZYMES AND COENZYMES.

Introduction
The use of enzymes in the diagnosis of disease is one of the important benefits
derived from the intensive research in biochemistry since the 1940's. Enzymes have
provided the basis for the field of clinical chemistry.

It is, however, only within the recent past few decades that interest in diagnostic
enzymology has multiplied. Many methods currently on record in the literature are
not in wide use, and there are still large areas of medical research in which the
diagnostic potential of enzyme reactions has not been explored at all.

Early Enzyme Discoveries

The existence of enzymes has been known for well over a century. Some of the earliest
studies were performed in 1835 by the Swedish chemist Jon Jakob Berzelius who termed
their chemical action catalytic. It was not until 1926, however, that the first enzyme was
obtained in pure form, a feat accomplished by James B. Sumner of Cornell University.
Sumner was able to isolate and crystallize the enzyme urease from the jack bean. His
work was to earn him the 1947 Nobel Prize.

John H. Northrop and Wendell M. Stanley of the Rockefeller Institute for Medical Research
shared the 1947 Nobel Prize with Sumner. They discovered a complex procedure for
isolating pepsin. This precipitation technique devised by Northrop and Stanley has been
used to crystallize several enzymes.

Definition.
Enzymes are often referred to as biological catalyst that speeds up the rate of chemical
reactions by converting substrate(s) to product(s).
N.B Not all enzymes are protein because we have ribozyme that is nucleic acid in nature.
Almost all processes in biological cells needs enzyme action(s) in order to occur at
significant of appreciable rate.

Naming and Classification

Except for some of the originally studied enzymes such as pepsin, rennin, and trypsin,
most enzyme names end in "ase". The International Union of Biochemistry (I.U.B.)
initiated standards of enzyme nomenclature which recommend that enzyme names
indicate both the substrate acted upon and the type of reaction catalyzed. Under this
system, the enzyme uricase is called urate: O2 oxidoreductase, while the enzyme
glutamic oxaloacetic transaminase (GOT) is called L-aspartate: 2-oxoglutarate
aminotransferase.

Enzymes can be classified by the kind of chemical reaction catalyzed.

 1. Addition or removal of water
A. Hydrolases - these include esterases, carbohydrases, nucleases,
deaminases, amidases, and proteases
B. Hydrases such as fumarase, enolase, aconitase and carbonic
anhydrase
2. Transfer of electrons
A. Oxidases
B. Dehydrogenases
3. Transfer of a radical
A. Transglycosidases - of monosaccharides
B. Transphosphorylases and phosphomutases - of a phosphate group
C. Transaminases - of amino group
D. Transmethylases - of a methyl group
E. Transacetylases - of an acetyl group
4. Splitting or forming a C-C bond
A. Desmolases
5. Changing geometry or structure of a molecule
A. Isomerases
6. Joining two molecules through hydrolysis of pyrophosphate bond in ATP or
other tri-phosphate
A. Ligases

Many enzymes are named by adding the suffix ‘-ase’ to the name of their substrate e.g
urease catalysis the hydrolysis of urea. However, this is not always true for all enzymes
e.g pepsin, trypsin acts on protein. The classification based on the International Union of
Biochemistry is broadly into six (6) classes thus:

CLASS1- OXIDOREDUCTASE- This group of enzyme catalyze the oxidation of one

substrate with simultaneous reduction of another substrate .e.g alcohol dehydrogenase

CLASS 2- TRANSFERASE- They catalyze the transfer of functional group(s) other

than hydrogen from one substrate to another; e.g hexose-6-phosphate transferase

CLASS 3- HYDROLASE- This class of enzyme hydrolyse ester, ether, peptide or

glycosidic bonds by adding water and the breaks the bond, e.g acetylcholine hydrolase

CLASS 4- LYASE: This group of enzyme removes group from a particular substrate or
breaks bonds by mechanism other than hydrolysis, e.g aldolase
CLASS 5- ISOMERASE-They catalyse the conversion of one isomer to the other. They
produce optical geometric or positional isomer of substrate, e.g triose phosphate
isomerase.

CLASS 6- LIGASE- This group catalyses the linkage of two substrates together, usually
with the simultaneous hydrolysis of ATP, e.g acetyl CoA carboxylase.

GENERAL PROPERTIES OF ENZYME.

(viii) Enzymes have enormous catalytic power i.e they can accelerate
reaction rate by at least a million
(ix) Enzymes are highly specific i.e highly specific both in the choice of
substrate and in reaction catalysed
(x) Activities of some enzymes are regulated i.e different kind of
regulatory mechanisms affect enzyme catalysed reaction.
(xi) Enzymes do not alter the reaction equilibria i.e enzymes do not alter
the equilibrium position, meaning that they accelerates the forward and
back ward reactions by precisely the same factor.
(xii) Enzymes decrease the activation energy e.g the lowers the activation
energy by reducing the transition state / activation complex
(xiii) Enzymes transform different kinds of energy i.e energy of reactant
could be converted into different form with high efficiency.

Chemical Nature of Enzymes

All known enzymes are proteins. They are high molecular weight compounds made
up principally of chains of amino acids linked together by peptide bonds. See Figure
1.

Enzymes can be denatured and precipitated with salts, solvents and other reagents.
They have molecular weights ranging from 10,000 to 2,000,000.
Many enzymes require the presence of other compounds - cofactors - before their
catalytic activity can be exerted. This entire active complex is referred to as the
holoenzyme; i.e., apoenzyme (protein portion) plus the cofactor (coenzyme,
prosthetic group or metal-ion-activator) is called the holoenzyme.

Apoenzyme + Cofactor = Holoenzyme

According to Holum, the cofactor may be:

1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable

and loosely attached to the protein part.

2. A prosthetic group - an organic substance which is dialyzable and thermostable

which is firmly attached to the protein or apoenzyme portion.

3. A metal-ion-activator - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++,
Mg++, Ca++, and Mo+++.

FACTORS AFFECTING THE RATE OF ENZYME CATALYSIS

Factors affecting the rate of enzyme catalyzed reactions include among others:
(i) temperature
(ii) pH
(iii) Substrate concentration
(iv) Presence or absence of activator(s) and/or inhibitor(s)
Temperature: The rate of an enzyme reaction varies with temperature according to the
Arrhenius equation i.e rate=Ae (-E/RT). The equation explains the sensitivity of enzyme
to temperature because of the relationship between the rate and temperature is
exponential. Each enzyme has optimum temperature after which is starts to denature

pH: The state of ionization of amino residues in the active site of an enzyme is pH
dependent. A typical enzyme has an optimum pH of activity.

Effect of substrate concentration: At constant enzyme concentration, when the

sudstrate concentration is low, the rate of reaction is very low. However, this increases
with an increase in substrate concentration. Later, a point will be reached beyond which
further increase in substrate concentration will not produce significant increase in
reaction velocity.

Influence of inhibitor /activator: Enzyme inhibitors combine specifically with an

enzyme to reduce its ability to convert substrate to products while activator enhances the
rate of an enzyme catalyzed reaction. There are two types of inhibitors namely:
(i) reversible inhibitor-which binds with non-covalent bonds
(ii) irreversible inhibitor-which bind with covalent bonds.
Reversible inhibitors are further divided into:
(i) competitive inhibitor i.e the one that competes with the substrate for binding
at the active site
(ii) non-competitive inhibitor i.e the one that binds at some other site apart from
the active site of the enzyme.
(iii) Uncompetitive inhibitor i.e the one that did not bind to the enzyme but only
bind to the enzyme –substrate (ES) complex..

Active site of an enzyme.

The active sits of an enzyme is that region of the enzyme where catalysis takes place. It is
also the region that binds the substrate and contributes the residues that directly
participates in the making and breaking of bonds.

Some features of active site are:

(i) it is a relatively small portion of the total enzyme volume
(ii) it is a three dimensional entity
(iii) substrate binds with relatively weak forces
(iv) it is a cleft or crevice
(v) the specificity of binding depends on the precisely defined arrangement of
atom in an active site

Note that: the interaction of substrate and enzyme could be expressed in term of two
models namel:
(i) lock and key model
(ii) induced fit model.

Enzyme Kinetics
Michaelis and Menten derived equation for enzyme catalyzed reaction involing a single
substrate and single product thus:
S→P
as
v =Vmax X [S]/Km +[S]
where v = initial velocity
Vmax = maximum velocity
[S] = substrate concentration
 Km = Michealis-Menten constant.
Note that: any enzyme that obeys M-M equation will give an hyperbolic curve when the
plot of v vs [S] is made.

Significance of Km and Vmax.

Km is the substrate concentration at half the maximum velocity. It is a measure of affinity
of an enzyme for substrate i.e the higher the Km the lower the affinity and vice versa.
Vmax is used to express the efficiency of an enzyme operation i.e often used to compare
the catalytic efficiency of different enzyme.

ALLOSTERIC ENZYMES
These are regulatory enzymes that functions through reversible non-covalent binding of a
modulatory molecule. They usually determine the rate of overall sequence of reaction
because they catalyze the committed/slowest step. Such enzyme is usually the first in the
sequence of a multienzyme reaction system. They are known to have the following
properties:
(i) They have both catalytic and regulatory sites for binding of substrate
(ii) Generally larger and more complex than the simple enzyme
(iii) Shows deviation from classical M-M behaviour in that thet give sigmodial
curve for the plot of v vs [S].
(iv) They undergo conformational changes in binding of modulatory molecule
(v) They may be inhibited by their modulator (-ve modulator) or stimulated by
modulator (+ve modulator)

CO-ENZYMES.

These are additional non-protein part of an enzyme that is required for enzymatic
activities. Inorganic forms of coenzyme are called cofactors. Tightly forms of coenzyme
are called prosthetic group.

The role of a cofactor is either:

(i) to alter the three-dimensional structure of the protein and/or the bound
substrate in order to activate the interaction of the enzyme with its substrate
(ii) to actually participate in overall reaction as another substrate.

Different types of coenzymes, type of reaction and group transfer are given below

COENZYMES TYPE OF REACTION GROUP TRANSFER

NAD+/NADP+ oxidation-reduction hydrogen (electron)

FAD, FMN oxidation-reduction hydrogen (electron)
Coenzyme A activation and transfer of acyl group acyl group
Lipoic acid acyl group transfer acyl group
Thiamine pyrophosphate acyl group transfer acyl group
Biotin carbon (iv) oxide fixation carbon (iv) oxide
Pyridoxal phosphate transamination amide (–NH2)
Tetrahydrofolic acid metabolism of one carbon fragment -CH3, -CH2