ENZYME BIOCHEMISTRY

ENZYME-BIOCHEMISTRY
Dr. NARESH PANIGRAHI
ASST.PROFESSOR
GITAM INSTITUTE OF PHARMACY
GITAM UNIVERSITY
VISAKHAPATNAM

ENZYMES
• Enzymes are the body's catalysts.
• Without them, the cell's chemical reactions
would be too slow and many would not occur at
all.
• A catalyst is an agent which speeds up a chemical
reaction without being changed itself.
• As an example, let us consider the substrate for
lactate dehydrogenase—an enzyme which
catalyses the reduction of pyruvic acid to lactic
acid

• They increase the rate of a reaction by
lowering the activation energy barrier.

How do catalysts lower activation energies?
• There are several factors at work.
Catalysts provide a reaction surface or
environment.
 Catalysts bring reactants together.
 Catalysts position reactants correctly so that they
easily attain their transition state configurations.
 Catalysts weaken bonds.
Catalysts may participate in the mechanism.

The Mechanism of Enzymatic Action

The reactant usually binds to the enzyme
forming a transition complex that stabilizes
the transition state.
• The interaction between enzyme and
substrate is very specific. Specificity may be
for a class of compounds or for a particular
compound. Eg. Hexokinase / glucokinase

Enzyme Structure
• All known enzymes are proteins.
• They are high molecular weight compounds made up
principally of chains of amino acids linked together by
peptide bonds.
• Many enzymes require the presence of cofactors
• Coenzyme= a non-protein organic substance which is
loosely attached to the protein part.(NAD+, NADH, FAD)
• Prosthetic group = an organic substance which is firmly
attached to the protein or apoenzyme portion or
• Metal-ion activators ( K+, Fe++, Fe+++, Cu++, Co++,
Zn++, Mn++, Mg++, Ca++)

The structure of an ENZYME CONTAINS
• AN Apoenzyme: Protein part
• Cofactor: Non-protein component
– Coenzyme: Organic cofactor
• Holoenzyme: Apoenzyme plus cofactor

Specificity of Enzymes
• One of the properties of enzymes that makes them so important as
diagnostic and research tools is the specificity they exhibit relative to
the reactions they catalyze.
• In general, there are four distinct types of specificity:
1. Absolute specificity - the enzyme will catalyze only one reaction.
2. Group specificity - the enzyme will act only on molecules that have
specific functional groups, such as amino, phosphate and methyl
groups.
3. Linkage specificity - the enzyme will act on a particular type of
chemical bond regardless of the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular steric
or optical isomer.

• In the early days, the enzymes were given names by
their discoverers in an arbitrary manner. For example,
the names pepsin, trypsin and chymotrypsin convey no
information about the function of the enzyme or the
nature of the substrate on which they act.
• Sometimes, the suffix-ase was added to the substrate
for naming the enzymes e.g. lipase acts on lipids;
nuclease on nucleic acids; lactase on lactose.
• These are known as trivial names of the enzymes
which, however, fail to give complete information of
enzyme reaction (type of reaction, cofactor
requirement etc.)
Nomenclature and
Enzyme Classification

Enzyme nomenclature
EC nomenclature
* each enzyme is classified by EC number (Enzyme
Commission of IUBMB) – 6 classes:
• EC 1.x.x.x oxidoreductases
• EC 2.x.x.x transferases
• EC 3.x.x.x hydrolases
• EC 4.x.x.x lyases
• EC 5.x.x.x isomerases
• EC 6.x.x.x ligases (synthetases)
→ classification by a reaction catalyzed by the enzyme

systematic names
* are made according to a special rules, they specify a
reaction catalyzed by the enzyme
example:
ATP : D-glucose phosphotransferase (EC 2.7.1.2)
 transfers (2) phosphate (7) to an alcohol group (1)
ATP + D-Glc  ADP + D-Glc-6-phosphate
(Glc-6-P)

common names (= accepted names)
* easier than the systematic names, widely used
* very important!
example:
EC 2.7.1.2. = glucokinase (see above)

Nomenclature and
Enzyme Classification
• There are 6 main classes based on the type of
reaction catalyzed.
• Oxidoreductase: Oxidation-reduction reactions
• Transferase: Transfer functional groups
• Hydrolase: Hydrolysis
• Lyase: Removal of atoms without hydrolysis
• Isomerase: Rearrangement of atoms
• Ligase: Joining of molecules, uses ATP

THE ACTIVE SITE OF AN ENZYME
• ACTIVE SITE: IS a Pocket in the enzyme where
substrates bind and catalytic reaction occurs.
• Active site is a relatively small 3-D region within
the enzyme.
• Substrates bind in active site by weak non-
covalent interactions
• A. hydrogen bonding
• B. hydrophobic interactions
• C. ionic interactions

THE ACTIVE SITE OF AN ENZYME
• The amino acids present in the active site play
an important role in enzyme function.
• Amino acids present in the active site can have
one of two roles.
1. Binding—the amino acid residue is involved in
binding the substrate to the active site.
2. Catalytic—the amino acid is involved in the
mechanism of the reaction.

Allosteric Enzymes
• Allosteric enzymes have one or more allosteric sites
• Allosteric sites are binding sites distinct from an
enzyme’s active site or substrate-binding site
• Molecules that bind to allosteric sites are called
effectors or modulators

Binding to allosteric sites alters the activity of
the enzyme. This is called cooperative binding.
Effectors may be positive or negative
Effectors may be homotropic or heterotropic
Regulatory enzymes of metabolic pathways
are allosteric enzymes (eg: feedback
inhibition)

Factors affecting Enzymes
• substrate concentration
• pH
• temperature
• inhibitors
© 2007 Paul Billiet ODWS

Substrate concentration: Non-enzymic reactions
• The increase in velocity is proportional to the
substrate concentration
Reaction
velocity
Substrate concentration

Substrate concentration: Enzymic reactions
• Faster reaction but it reaches a saturation point when all the
enzyme molecules are occupied.
• If you alter the concentration of the enzyme then Vmax will
change too.
Reaction
velocity
Substrate concentration
Vmax

The effect of pH
Optimum pH values
Enzyme
activity Trypsin
Pepsin
pH
1 3 5 7 9 11

The effect of pH
• Extreme pH levels will produce denaturation
• The structure of the enzyme is changed
• The active site is distorted and the substrate
molecules will no longer fit in it
• At pH values slightly different from the enzyme’s
optimum value, small changes in the charges of the
enzyme and it’s substrate molecules will occur
• This change in ionisation will affect the binding of the
substrate with the active site.

The effect of temperature
• Q10 (the temperature coefficient) = the increase in
reaction rate with a 10°C rise in temperature.
• For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every
10°C rise in temperature)
• Enzyme-controlled reactions follow this rule as they
are chemical reactions
• BUT at high temperatures proteins denature
• The optimum temperature for an enzyme controlled
reaction will be a balance between the Q10 and
denaturation.

Temperature / °C
Enzyme
activity
0 10 20 30 40 50
Q10 Denaturation

• For most enzymes the optimum temperature is
about 30°C
• Many are a lot lower,
cold water fish will die at 30°C because their
enzymes denature
• A few bacteria have enzymes that can withstand very
high temperatures up to 100°C
• Most enzymes however are fully denatured at 70°C

Inhibitors
• Inhibitors are chemicals that reduce the rate
of enzymic reactions.
• The are usually specific and they work at low
concentrations.
• They block the enzyme but they do not usually
destroy it.
• Many drugs and poisons are inhibitors of
enzymes in the nervous system.

The effect of enzyme inhibition
• Irreversible inhibitors: Combine with the
functional groups of the amino acids in the
active site, irreversibly.
Examples: nerve gases and pesticides,
containing organophosphorus, combine with
serine residues in the enzyme acetylcholine
esterase.

• Reversible inhibitors: These can be washed
out of the solution of enzyme by dialysis.
There are two categories.

1. Competitive: These
compete with the
substrate molecules for
the active site.
The inhibitor’s action is
proportional to its
concentration.
Resembles the substrate’s
structure closely.
Enzyme inhibitor
complex
Reversible
reaction
E + I EI

Succinate Fumarate + 2H++ 2e-
Succinate dehydrogenase
CH2COOH
CH2COOH CHCOOH
CHCOOH
COOH
COOH
CH2
Malonate

Enzyme Inhibitors: Competitive
Inhibition

2. Non-competitive: These are not influenced by the
concentration of the substrate. It inhibits by binding
irreversibly to the enzyme but not at the active
site.
Examples
• Cyanide combines with the Iron in the enzymes
cytochrome oxidase.
• Heavy metals, Ag or Hg, combine with –SH groups.
These can be removed by using a chelating agent such
as EDTA.

Applications of inhibitors
• Negative feedback: end point or end product
inhibition
• Poisons snake bite, plant alkaloids and nerve
gases.
• Medicine antibiotics, sulphonamides,
sedatives and stimulants

Enzyme Inhibitors: Noncompetitive
Inhibition

Enzyme inhibitors in Medicine
• The effectiveness of an enzyme inhibitor as a
therapeutic agent will depend on
(1) The potency of the inhibitor,
(2) Its specificity toward its target enzyme,
(3)The choice of metabolic pathway targeted for
disruption, and
(4) The inhibitor or a derivative possessing
appropriate pharmacokinetic characteristics.

Coenzyme
• The protein part of the enzyme, on its own, is
not always adequate to bring about the
catalytic activity.
• Many enzymes require certain non-protein
small additional factors, collectively referred
to as cofactors for catalysis.
• The cofactors may be organic or inorganic in
nature.

Cofactor
• 1. Prosthetic group (when cofactor is very
tightly bound to the apoenzyme and has small
size )
• 2. Metal ion
• 3. Coenzyme(organic molecule derived from
the vitamin-B which participate directly in
enzymatic reactions)

Defination
• The non-protein, organic, Iow molecular weight
and dialysable substance associated with enzyme
function is known as coenzyme.
• The functional enzyme is referred to as
holoenzyme which is made up of a protein part
(apoenzyme) and a non-protein part (coenzyme);
• The term prosthetic group is used when a non-
protein moiety is tightly bound to the enzyme
which is not easily separable by dialysis.
• The term activator is referred to the inorganic
cofactor (like Ca2+, Mg2+, Mn2+ etc.; necessary
to enhance enzyme activity

Functions of coenzymes
• Function of apoenzyme:
• It is responsible for the reaction
• Function of cofactor:
• It is responsible for the bonds formation between enzyme
and substrate
• Transfer of functional groups
• Takes place in the formation of tertiary structure of protein
part.
• Coenzymes participate in various reactions involving transfer
of atoms or groups like hydrogen, aldehyde, keto, amino,
acyl, methyl, carbon dioxide etc.
• Coenzymes play a decisive role in enzyme function

Prosthetic group
• 1. Heme group of cytochromes
• 2. Biothin group of acetyl-CoA carboxylase

Metal ions
• Fe - cytochrome oxidase, catalase
• Cu - cytochrome oxidase, catalase
• Zn - alcohol dehydrogenase
• Mg - hexokinase, glucose-6-phosphatase
• K, Mg - pyruvate kinase
• Na, K – ATP-ase

Coenzyme
• B1
• TPP- Thiamine Pyro Phosphate
• B2
• FAD- Flavin Adenine Dinucleotide
• FMN- Flavin Mono Nucleotide
• Pantothenic acid
• Coenzyme A (CoA)
• B5
• NAD – Nicotinamide Adenine Dinucleotide
• NADP- Nicotinamide Adenine Dinucleotide
Phosphate

Nucleotide coenzymes
Some of the coenzymes possess
nitrogenous base, sugars and
phosphate. Such coenzymes are,
therefore, regarded as nucleotides
e.g. NAD+, NADP+, FMN, FAD,
coenzyme A, UDPC etc

Coenzymes do not decide enzyme
specificity
• A particular coenzyme may participate in catalytic
reactions along with different enzymes.
• For instance, NAD+ acts as a coenzyme for lactate
dehydrogenase and alcohol dehydrogenase.
• In both the enzymatic reactions, NAD+ is involved
in hydrogen transfer.
• The specificity of the enzyme is mostly
dependent on the apoenzyme and not on the
coenzyme.

Applications of enzymes
• Certain enzymes are useful as therapeutic
agents, analytical reagents, manipulations in
genetic and for industrial application.

Isoenzymes (isozymes)
• Isoenzymes (isozymes) are enzymes which catalyze
the same reaction but differ in their primary
structure and physico chemical properties.
• which include the structure, electrophoretic and
immunological properties,
• Km and Vmax values, pH optimum,
• Relative susceptibility to inhibitors and
• degree of denaturation.

• Isoenzymes are
• produced by different genes (= true isozymes)
• or produced by different post translational modification
(= isoforms)
• found in different compartments of a cell
• found in different tissues of an organism
• can be oligomers of various subunits (monomers)

Explanation for the
existence of isoenzymes
• Many possible reasons are offered to explain
the presence of isoenzymes in the living
systems.
1. lsoenzymes synthesized from different genes
e.g. malate dehydrogenase of cytosol is
different from that found in mitochondria.
2. Oligomeric enzymes consisting of more than
one type of subunits e.g. lactate
dehydrogenase and creatine phosphokinase.

3. An enzyme may be active as monomer or
oligomer e.g. glutamate dehydrogenase.
4. In glycoprotein enzymes, differences in
carbohydrate content may be responsible for
isoenzymese .g. alkaline phosphatase.

S. No Property E.g.
1 Electrophoretic
mobility
Isoenzymes of Lactate dehydrogenase have
different electrophoretic mobility
2 Heat stability Alkaline phosphatase isoenzymes are either heat
labile or stable
3 Inhibitor An inhibitor can inhibit only one isoenzyme of an
enzyme eg. Acid phosphatase
4 Km Glucokinase and hexokinase
5 Cofactors Mitochondrial isocitrate dehydrogenase requires
NAD+ , cytosolic form requires NADP+
6 Tissue localisation LDH 1 is present in heart, LDH 5 in muscle
7 Antibodies For creatine kinase, each isoenzyme can be bound
only by a specific antibody

Uses of isoenzymes
• Some enzymes are specific to particular tissue.
• Normally they are not seen in blood or seen in
negligible levels.
• Usually when a tissue dies, it gets necrosed
and the enzymes inside the cells of the tissues
gets released in the circulation
• Measuring the levels of those enzymes aids in
identification of the organ that got damaged.

lsoenzymes of lactate dehydrogenase
(LDH)
• LDH whose systematic name is L-lactate- NAD+
oxidoreductase (E.C. 1 .'1.1.27) catalyses the
interconversion of lactate and pyruvate as
shown below

Lactate dehydrogenase
• It occurs in 5 possible forms in the blood
serum:
• LDH1
• LDH2
• LDH3
• LDH4
• LDH5
They can be separated by electrophoresis
(cellulose or starch gel or agarose gel).
LDHI has more positive charge and fastest in
electrophoretic mobility while LDH5 is the slowest.

Structure of LDH isoenzymes
• LDH is an oligomeric (tetrameric) enzyme made
up of four polypeptide subunits.
• Two types of subunits namely M (for muscle) and
H (for heart) are produced by different genes.
• M-subunit is basic while H subunit is, acidic.
• The isoenzymes contain either one or both the
subunits giving LDHI to LDH5. The characteristic
features of LDH isoenzymes are given as below

Structure of LDH
• Each contains 4 polypeptide chains which are
of 2 types: A and B which are usually called M
(muscle) and H (heart).
• LDH1 –H H H H
• LDH2 – H H H M
• LDH3 – H H M M
• LDH4 – H M M M
• LDH5 – M M M M

Significance Of Differential Catalytic Activity :
• LDHl (Ha) is predominantly found in heart muscle and is
inhibited by pyruvate – the substrate. Hence, pyruvate is
not converted to lactate in cardiac muscle but is converted
to acetyl CoA which enters citric acid cycle.
• LDH5 (M+) is mostly present in skeletal muscle and the
inhibition of this enzyme by pyruvate is minimal , hence
pyruvate is converted to lactate.
• Further, LDH5 has low K. (high affinity) while LDHl has high
Km (low affinity) for pyruvate.
• The differential catalytic ativities of LDHl and LDH5 in heart
and skeletal muscle, respectively, are well suited for the
aerobic (presence of oxygen) and anaerobic (absence of
oxygen) conditions, prevailing in these tissues.

Clinical importance of LDH
• Acute myocardial infarction
• LDH1 and LDH2
• Acute liver damage
• LDH4 and LDH5

ENZYME BIOCHEMISTRY

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