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MBC 302: BASIC ENZYMOLOGY & IMMUNOCHEMISTRY 3 UNITS; C
Mr Kweki
Classification and nomenclature of enzymes. Mechanisms of enzymes-catalyzed reactions. Effects
of temperature, (pH), ions and inhibitors on enzymes. Active sites of enzymes. Concepts of
‘cooperativity. Estimation of kinetic parameters — enzymes activities, Km, Vmax, Ki, et.
Miss Vanessa
Isoenzymes, zymogen activation, digestive enzymes. Production, isolation, purification and
characterization of enzymes from animal and plant tissues. Diagnostic enzymes.
Prof Mordi
Concept and types of immunity. Immunogens, antigens and haptens. Biochemical requirements.
for immunogenicity. Antibodies; class, structure, synthesis and function. Antigen-antibody
reactions. The biochemistry of hypersensitivity and allergy. Major histocompatibility complex
and immunological tolerance
Dr Orororo
Autoimmunity and concept of autoantibodies. Biochemistry of the complement system.
Applications of immune-biochemisty in clinical and biological studies; production of specific
antibody. Radio-immuno assay (RIA) and blood group serology. An outline of Immuno-
diffusion techniques.UNIT ONE: NATURE OF ENZYMES
WHAT ARE ENZYMES
Enzymes are biological catalysts. They increase the rate of chemical reactions taking place within
living cells without themselves suffering any overall change. The reactants of enzyme-catalyzed
reactions are termed substrates. Each enzyme is quite specific in character, acting on a particular
substrate or substrates to produce a particular product or products. All enzymes are proteins.
However, without the presence of a non-protein component called a cofactor, many enzyme
proteins lack catalytic activity. When this isthe case, the inactive protein component of an enzzyme
is termed the apoenzyme, and the active enzyme, including cofactor, the holoenzyme. The
cofactor may be anorganic molecule, when it is known as a coenzyme, or it may be a metal ion.
Some enzymes bind cofactors more tightly than others. When a cofactor is bound so tightly that it
is difficult to remove without damaging the enzyme, itis sometimes called a prosthetic group.
To summarize diagrammatically
oo Simple Enzyme
oe ~ Apoenzyme
Holo Enzyme a
aN. Cofactor
A BRIEF HISTORY OF ENZYMES.
Unvil the nineteenth century, it was considered that processes such as the souring of mill and the
fermentation of sugar to alcohol could only take place through the action of a livitlg organism. in
1833, the active agent breaking down the sugar was partially isolated and given the name diastase
(now known as amylase). A litte later, a substance which digested dietary protein was extracted
from gastric juice and called pepsin. These and other active preparations were given the generaltame ferments, Justus von Liebig recognized that these ferments could be non-living materials
obtained from living cells, but Louis Pasteur and others still maintained that ferments must contain
living material,
|While this dispute continued, the term ferment was gradually replaced by the name enzyme. This
\was first proposedby Wilhelm Kfthne in 1878, and comes from the Greek, enzume meaning ‘in
‘yeas’, Appropriately, it was in yeast that a factor was discovered which settled the argument in
favour of the inanimate theory of catalysis: brothers Eduard and Hans Buchner showed, in 1897,
that sugar fermentation could take place when a yeast cell extract was added even though no living
cells were present. In 1926, James Sumner erystallized urease from jack-bean extracts and, in the
next few years, many other enzymes were purified and crystallized. Once pure enzymes were
available, their structure and properties could be determined, and the findings form the material
for most ofthis book. Today, enzymes still form a major subject for academic research. They are
investigated in hospitals as an aid to diagnosis and, because of their specificity of action, are of
great value as analytical reagents. Enzymes are still widely used in industry, continuing and
‘extending many processes which have been used since the
dawn of history.
‘THE NAMING OF ENZYMES
‘There is long tradition of giving enzymes names ending in '-ase’. The only major exceptions to
this are the proteolytic enzymes, ie. ones involved in the breakdown of proteins, whose names
usually end with '-in', e.. trypsin. The names of enzymes usually indicate the substrate involved.
Thus, lactase catalyses the hydrolysis of the disaccharide lactose to its component
‘monosaccharides, glucose and galactose:
Ci HnOn +20. <———* Cai20s + CHO
‘The name lactase is a contraction of the clumsy, but more precise, lactosase. The former is used!
because it sounds better but it introduces a possible trap for the unwary because it could exsily
suggest an enzyme acting on the substrate lactate. There is nothing in the name of this enzyme oF
2many others to indicate the type of reaction being catalysed. Fumarase, for example, by analogy
with lactase might be supposed to catalyse a hydrolytic reaction, but, in fact, it hydrates fumarate
to form malate:
‘The names of other enzymes, e.g. transearboxylase, indicate the nature of the reaction without
specifying the substrates (which in the case of transcarboxylase are methylmalonyl-CoA and
pyruvate). Some names, such as eatalase, indicate neither the substrate nor the reaction (catalase
‘mediates the decomposition of hydrogen
peroxide). Needless to say, whenever a new enzyme has been characterized, great care has usually
been taken not to give it exactly the same name as an enzyme catalyzing a different reaction. Also,
the names of many enzymes make clear the substrate and the nature of the reaction being catalyzed.
For example, theres little ambiguity about the reaction catalyzed by malate dehydrogenase. This
‘enzyme mediates the removal of hydrogen from malate to produce oxaloacetate:
However, malate dehydrogenase, like many other enzymes, has been known by more than one
rname. So, because ofthe lack of consistency inthe nomenclature, it became apparent as the list of
known enzymes rapidly grew that there was a need for a systematic way of naming and classifying
enzymes. A commission was appointed by the International Union of Biochemistry (later re-named
the Intemational Union of Biochemistry and Molecular Biology, IUBMB), and its report
‘Published in 1964, forms the basis of the currently accepted system. Revised editions of the report
‘were published in 1972, 1978, 1984 and 1992. An electronic version is now maintained by the
IUBMB on an accessible web-site, and this is updated on a regular bass.
‘The Enzyme Commission's system of classification
‘The Enzyme Commission divided enzymes into six main classes, on the basis of the total reaction
catalysed. The classification of enzyme was described in 1961 by enzyme commission of the
Intemational Union of
Biochemistry (TUB). According to this classification, each enzyme is characterized by « code
number called enzyme code number or EC’ number, consisting of four digits. According to the
TUB system, enzymes are classified into six major classes as followsClassification And Examples of Enzymes With Their Main Class
sino. | Enzyme | Class of Enzymes Subclasses of | Examples
code(EO) Enyzmes
T [ECT | Oxidoreductases Dehydrogenases | Lactate dehydrogenase
Reductases pH)
Oxidases Glucose ‘6-phosphate
Peroxidases dehydrogenase
(G-6-PD)
Cytochrome oxidase
2 [EC-2 | Transferases ‘Amino transferase | Aspartate |
ortransaminase | aminotransaminase(AST)
Kinase Alanine aminotransaminase
Transcarboxylase | (ALT)
Hexokinase
3 [ECS | Hydrolases ‘Acid phosphatase | Lipase
All digestive | a-Amylase
enzymes like | Trypsin
amylase, pepsin, | Lactase
typsin, Sucrase |
chymotrypsin | Pepsin |6 [EC | Ligases. ‘Thiokinase Glutamine synthetase
DNA ligase Pyruvate carboxylase
Chelatases DNA ligases
Synthetase
Oxidoreductases
‘These enzymes catalyze oxidation-reduetion reactions which can be illustrated schematically as
follows ‘
A +) Be———+Bi+ a
(red) (0x) (red) (ox)
Specific Example
Nap" He
Pyruvate
‘Transferases
Enzymes that catalyze the transfer of amino, carboxyl, methyl or phosphoryl groups from one
molecule to another are called transferases. These reactions can be illustrated as follows
AY +B4+———ra + BY
Notable exampleALT
Alanine + a-Ketoglutarate === Pyruvate + Glutamate
PLP
where,
ALT:: Alanine aminotransferase and PLP : Pyridoxal phosphate (coenzyme)
Hydrolases
Enzymes of this class catalyze the cleavage of C-O, C-N, C-C and some other bonds with the
addition of water. These can be illustrated as follows
X-Y+HO «______+X-OH + BH
Notable Example
Glucose-6-phosphate + Hxo ————__—>> Glucose + Pi
Lyases
Lyases cleavage C-O, C-C, and C-N bonds via other means than hydrolysis or oxidat
nto
produce compounds with double bonds or catalyze the reverse reaction, by the addition of « group
to.a double bond. In cases where reverse reaction is important, then synthase, (not synthetase of
group EC-6) i used inthe name. This type of reaction is illustrated as follows:
atx ‘Wyases)
fy Siig) 5
+X-YCommon exampl
Fructose 1,6- <——> Glyceraldehyde- 3-phosphate «
bisphosphate Dihydroxy actone phosphate
Tsomerases
Isomerases are groups of enzymes that catalyze intramolecular structural rearrangement in &
molecule, They are called epimerases, isomerases, or mutases, depending on the (ype of
isomerism catalyzed. This reaction can be illustrated as follows:
ABC + CAB
‘Common example
Ligases (Synthetases)
Ligases are notable for the joining of two molecules coupled with the hydrolysis of ATP. The
reaction is illustrated as follows:
A+B+AIP > AB +ADP + Pi‘Common example
Glutamate + NHy + ATP > Glutamine + ADP * Pi
PROPERTIES OF ENZYME,
1) Active si
Enzyme molecules have an active site, o cleft, in which amino acid chains form a 3-limensional
surface that is complementary to the substrate, The active site binds to the substrate, and (he
enzyme substrate (ES) complex is converted into an enzyme-produet (EP); which then dissociate
into an enzyme and a product. Each enzyme has one ot more "active sites” where it can take
the substrate. These "active sites" can include free hydroxyl groups of serine and tyrosine, as well
asthe phenolic and sulfhydryl groups of SH-hiol and imidazole groups of cysteine and histidine
to interact with substrates. It is also possible that the active site (Catalytic sie) is different from
the binding site in which case they are situated closely together in the enzyme molec
2), Catalytic efficency/ Enzyme turnover number
Enzyme-catalyzed reactions are the most efficent, taking between 103 and 108 times
‘unaccelerated reactions. Fach enzyme molecule is usually able to convert between 100 and 1000
substrate molecules into a product every second. The enzyme tum over number is the numbe
substrate molecules converted in a given unit time (Carbonic Anhydrase being the fastest enzyme
3). Specificity of Enzyme Action.
Specificity refers to the ability of an enzyme to discriminate between two competing sub
Enzymes are highly specific both in the reaction catalyzed anSpecificity makes it possible for a number of enzymes to co-exist inthe cell without interfering in
‘each other's actions.
Types of Specificity
‘The following types of specificity have been recognized:
1. Substrate specificity
2. Reaction specificity
3. Stereo specificity.
1), Substrate Specificity
‘There ae the following types of substrate specificity
4). Absolute substrate specificity
Certain enzymes will act on only one substrate and catalyze one reaction, ¢. Glucokinase, lactase,
‘urease, ete.
Glucose ————> Glucose-6-phosphate
Lactose Glucose +Galactose
Ura > Ammonia + co,
»), Relative substrate specificity
In this case, the enzyme acts on more than one substrate. The relative substrate specificity is of
two types:
a. Group specificity
b. Bond specificity
1). Group specificity, the enzyme acts on more than one substrate containing a particular group,
ce
chymotrypsin acts on several proteins by hydrolyzing peptide bonds attached to arom
acids. Trypsin hydrolyzes peptide linkages involving arginine or lysine
ii), Bond specificity, here, the enzyme acts on more than one substrate containing « particulier
kind of bond, e.g. salivary a-amylase cleaves a-{I-4) glycosidic bonds of earbolydra
hydrolyzes ester bonds of lipidsBroad substrate specificity
an enzyme acts on more than one structurally related substrates, e-8, hexokinase
In this case,
catalyzes
the phosphorylation of more than one kind of hexoses such as glucose, fructose and mannose
2), Reaction Specificity
Inthis case, an enzyme is speci
‘one type of reaction. For example, pyruvate can undergo several reactions:
catalyzed by a separate enzyme, which catalyzes only that react and none other.
fic to a particular reaction but not to substrate (8) and catalyzes only
Each reaction is
Fig 9. Example of reaction specificity
3), Stereo Specificity
‘Numbers of enzymes show specificity towards stereoisomers, ic. they act on only one type of
isomer, For example, L-lactate dehydrogenase will act only on L-lactic acid and not D-lactic aid
Likewise, L-amino acid oxidase and D-amino acid oxidase are distinct enzymes which act only on
Land D-amino acids respectively. D-glucose oxidase can similarly act only on D- glucose and not
‘on L-glucose. Salivary a-amylase acts on the a-1,4 glycoside linkage and is inactive on b-I4
slycoside linkage. Isomerase and epimerase do not show stereospecificity
4, Regulation: Enzyme activity can be regulated that i, enzyme can be, activated or inhibited so
thatthe rate of product formation responds to the needs of the cel
5). Zymogens: These are inactive form of enzyme. Some enzymes are produced in mature in am
inactive form which can be activated when they are required. Such type of enzymes are called
‘Zymogens (Proenzymes).Many of the digestive enzymes and enzymes concemed with blood
‘coagulation are in this group. Examples: Pepsinogen - This zymogen is from gastric juice. Whem
required Pepsinogen converts to Pepsin. Trypsinogen - This zymogen is found in the puncrewtie
Juice, and when itis required gets converted to trypsin. The activation is brought about by specie
nions or by other enzymes that are proteolytic, Zymogen forms of enzymes a protective mechanism
to preven auto digestion ofssue producing the digestive enzymes and to prevent intravascular
coagulation of blood.
Isoenzymes (Isozymes Isoenzymes or isozymes are multiple forms isomers) of the same enzyme
action. Isoenzymes show different chemical and physical
that catalyze the same biochemical re
properties like electrophoreiie mobility and kinetic properties. Not all enzymes have isoenzyins
In fact, it was found that only those enzymes, which are active in polymeric form demonstrate
isoenzyme. For example:
1. Lactate dehydrogenase (LDH)
2, Creatine kinase (CK) (formerly called creatine
Alkaline
phosphokinase (CPK). other examples of isoenzymes are Acid phosphatase,
phosphatase, Amylase,
Hexokinase ete
Lactate Dehydrogenase (LDH)
Lactate dehydrogenase is a tetrameric enzyme that catalyzes the oxidation of L-lactae to pyruvate
It has five isoenzymes:
-LDHI
-LDH2
-LDH3
-LDHS
-LDHS.
LDH is a tetramer, made up of two types of polypeptide
(muscle) type and H (heart) type.
five
combinations are possible with varying ratios of two kinds of poly
LDH can be detected by electrophoresis as they have differen
is the fastest moving fraction towards the anode, it pr
Erythrocytes. However, LDHS is the least moving isoenz;
and in skeletal muscle.