[Link] [Link].

,Assistant Professor,
Department of Pharmaceutical Chemistry.
 Introduction, properties, nomenclature and IUB
classification of enzymes
 Enzyme kinetics (Michaelis plot, Line Weaver Burke plot)
 Enzyme inhibitors with examples
 Regulation of enzymes: enzyme induction and repression,
allosteric enzymes regulation
 Therapeutic and diagnostic applications of enzymes and
isoenzymes
 Coenzymes –Structure and biochemical functions
All living things require energy.
 Nutrients are one source of energy, as well as being
molecules organisms require
to grow, reproduce or repair.
 Biochemical reactions are the
processes used for the
formation, breakdown and
rearrangement of molecules to
provide organisms with energy.
 Activation Energy is the required input of energy to make
a reaction start
 A catalyst is a chemical that speeds up the reaction but is
not used up in the reaction
◦ Lowers the activation energy needed to start a reaction
◦ Is not used up during the reaction
◦ Is unchanged after a reaction
 Enzymes act as bio-catalyst.
 Enzymes are proteins that
speed up a rate of reaction
 Found in cells throughout the body
 Lowers activation energy
 Increasing the temperature make molecules move faster
 Biological systems are very sensitive to temperature
changes.
 Enzymes can increase the rate of reactions without
increasing the temperature.
 They do this by lowering the activation energy.
 They create a new reaction pathway “a short cut”
 Enzyme controlled reactions proceed 108 to 1011 times
faster than corresponding non-enzymic reactions.
The substrate
 The substrate of an enzyme are the reactants that are
activated by the enzyme
 Enzymes are specific to their substrates
 The specificity is determined by the active site
The active site
 One part of an enzyme, the active site, is particularly
important
 The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
more easily
Cofactors
 An additional non-protein molecule that is needed by some
enzymes to help the reaction
 Tightly bound cofactors are called prosthetic groups
 Cofactors that are bound and released easily are called
coenzymes
 Many vitamins are coenzymes
Specificity
1. Absolute Specificity – the characteristic that an enzyme
acts on only one substrate.
Eg: Glucokinase - Phosphorylates only glucose,
Urea is the only substrate for urease.
Specificity
2. Relative Specificity – the characteristic that an enzyme
acts on several structurally related substrates.
Eg: Hexokinase can catalyze phosphorylation of glucose,
galactose & mannose.
Specificity
3. Stereochemical Specificity – an enzyme's ability to
distinguish between stereoisomers
Eg: L-Amino acid oxidase – oxidises only L-amino
acids into α-keto acid and ammonia.
D-Amino acid oxidase – oxidises only D-amino acids
into α-keto acid and ammonia.
Denaturation - The process of unfolding
Most named for substrates & for reactions, with suffix “ase”
 The name of an enzyme in many cases end in –ase
For example, sucrase catalyzes the hydrolysis of sucrose
 The name describes the function of the enzyme
For example, oxidases catalyze oxidation reactions
 Sometimes common names are used, particularly for the
digestion enzymes such as pepsin and trypsin
 Some names describe both the substrate and the function
For example, alcohol dehydrogenase oxides ethanol
 EC 1. Oxidoreductases

 EC 2. Transferases
 EC 3. Hydrolases
 EC 4. Lyases
 EC 5. Isomerases
 EC 6. Ligases
 PRINCIPLE OF THE INTERNATIONAL
CLASSIFICATION

Each enzyme has classification number consisting of four

digits:

Example, EC: ([Link]) HEXOKINASE

EC: ([Link]) these components indicate the following
groups of enzymes:
 2. IS CLASS (TRANSFERASE)
 7. IS SUBCLASS (TRANSFER OF PHOSPHATE)
 1. IS SUB-SUB CLASS (ALCOHOL IS PHOSPHATE
ACCEPTOR)
 1. SPECIFIC NAME (Hexokinase)
ATP,D-HEXOSE-6 PHOSPHOTRANSFERASE
 1. Hexokinase catalyzes:

Glucose + ATP  glucose-6-P + ADP

6 CH2OH 6 CH OPO 2
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate
CLASSIFICATION
 MECHANISM OF ACTION
 Michaelis and Menten Concept
 When the enzyme and substrate are connected, it is
known as enzyme-substrate complex
 The binding site is where the enzyme physically attaches
itself to the substrate
 The active site is where the enzyme will cause a specific
part of the substrate to change
 MECHANISM OF ACTION
When a substrate (S) fits properly in an active site, an
enzyme-substrate (ES) complex is formed:
E + S  ES
THEORIES
 Fischer’s Lock and Key Model

+ S +
S
P

E + S ES complex E + P
The Induced Fit Hypothesis
 Some proteins can change their shape (conformation)
 When a substrate combines with an enzyme, it induces a
change in the enzyme’s conformation
 The active site is then moulded into a precise conformation
 Making the chemical environment suitable for the reaction
 The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)
 Koshland’s Induced Fit Model

P
S
S
P

E + S ES complex E + P
 Enzyme Concentration
 Substrate Concentration
 pH
 Temperature
 Inhibitors
 1. Enzyme Concentration
 The increase in velocity is proportional to the enzyme concentration

Reaction
velocity

Enzyme concentration
 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. Vmax

Reaction
velocity

Substrate concentration
 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.
 Optimum pH values

Enzyme
activity Trypsin

Pepsin

1 3 5 7 9 11
pH
 Q10 (the temperature coefficient) = the increase in
reaction rate with a 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.
 Q10 Denaturation
Enzyme
activity

0 10 20 30 40 50
Temperature / °C
 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 number of molecules of substrate with which a
single enzyme can react at a given time (ex.
reactions/minute) is known as the turnover number
◦ Can be quite large compared to uncatalyzed
reeactions
◦ Can depend on the environment
 Order of reaction : When the velocity of the reaction is
almost proportional to the substrate concentration( i.e. [S] is
less than Km), the rate of reaction is said to be first order
with respect to substrate. When the [S] is much greater than
Km, the rate of reaction is independent of substrate
concentration, and the reaction is said to be zero order.
 Enzyme kinetics and Km value : The enzyme (E) and
substrate (S) combine with each other to form an unstable
enzyme-substrate complex (ES) for the formation of
product (P).
The process of inhibition of enzymes activity.
1. Reversible Enzyme Inhibition:
a. Competitive Reversible Enzyme Inhibition
b. Noncompetitive Reversible Enzyme Inhibition
2. Irreversible Enzyme Inhibition
3. Allosteric Inhibition
 These can be washed out of the solution of enzyme by dialysis.
 There are two categories.
 a. Competitive - mimic the substrate and bind to the active site.
 b. Noncompetitive - bind to some other part of the enzyme.
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.
 Succinate Fumarate + 2H++ 2e-
Succinate dehydrogenase
CH2COOH CHCOOH

CH2COOH CHCOOH

COOH
COOH

CH2

COOH COOH
Oxalate Malonate
 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.
 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.
 Allosteric Enzyme – an enzyme with a quaternary structure
whose activity is changes by the binding of a modulator
 Cell’s can’t have enzymes turned on all the time
 The control of an enzyme complex by the binding of a
regulatory molecule.
 Regulatory molecule may stimulate or inhibit the enzyme
complex by causing it to change shape.
 Works like a reversible non-competitive inhibitor
Allosteric regulation
Heterotropic ligand binding modulates substrate binding and
catalysis.
Feedback Regulation
Homotropic regulation – Multisubunit
Covalent modification – Reversible
Phosphorylation, nucleotides, lipid anchors
 The end product of a metabolic pathway affects the function
of an enzyme
A metabolic pathway is switched off by the inhibitory
binding of its end product to an enzyme that acts early in
the pathway
 Usually allosteric regulation
 The enzyme without its non protein moiety is termed as
apoenzyme and it is inactive.
 Holoenzyme is an active enzyme with its non protein
component.
 Some enzymes need an additional molecule like co-factors /
co-enzymes to carry out the process.
Cofactors are molecules that serve an enzyme helpers.
A cofactor can be a metal ion or an organic molecule.
A cofactor that is an organic molecule is called a
coenzyme.
Vitamin derived Co-enzymes
I. Enzymes as Therapeutic Agents (Drugs)

S. Enzyme Disease/therapy
No
1 Streptokinase Clot lysis in myocardial infarction,
trauma, bleedings
2 Aspariginase Acute lymphocytic leukemia

3 Adenosine Severe combined immuno-deficiency

deaminase
syndrome (SCID)
 Enzymes as Drug Targets
Enzyme targeting Drug
Dihydrofolate reductase Antifolates: methrotrexate (cancer)
pyrimethamine (protozoa, malaria)
trimethoprim (bacteria)
Xanthine oxidase Allopurinol (hyperuricemia, gout)
(Purine metabolism)
Thymidylate synthase 5-Fluorouracil &
(Pyrimidine metabolism) 5-fluorodeoxyuridine (cancer)
Glycopeptide transpeptidase Antibiotics, penicillin
HIV-Reverse transcriptase 3’-azido-2’,3’-dideoxythymidine (AZT)
HIV & SARS proteases Ritonavir, saquinavir (clinical trial phase)
 II. Enzymes as Diagnostic Agents
Enzyme Cause of elevated plasma level
Acid phosphatase - ACP Prostatic cancer
Alkaline phosphatase – ALP Rickets, hypoparathyroidism, osteomalacia,
obstructive jaundice, cancer of bone/liver
Alanine aminotransferase – ALT Hepatitis, jaundice, circulatory faillure
Aspartate aminotransferase – AST Myocardial infarction, muscle damage, anemia,
hepatitis, circulatory faillure with liver congestion
Amylase - AM Acute pancreatitis, peptic ulcer
-Glutamyl transferase – GMT Hepatitis, alcoholic liver damage, cholestasis
Creatine kinase – CK Myocardial infarction, Muscular dystrophy
Lactate dehydrogenase – LD1 > LD2Myocardial infarction, kidney disease,
LD2, LD3 Leukemia
LD5 Liver disease, muscle damage
 ENZYMES - USED IN LABORATORY ASSAYS
Components of commercial kits or diagnostic strips
- determination of glucose - glucose oxidase, peroxidase
- determination of cholesterol - cholesterol esterase,
cholesterol oxidase, peroxidase,
- determination of urea – urease,
Markes in the immunochemical analysis
- ELISA (=enzyme-linked immunoadsorbent assay) –
peroxidase, alkaline phosphatase.
 III. Industrial Applications
Enzyme Application
Renin Cheese Preparation
Glucose isomerase Production of high fructose syrup
α-Amylase In food industry to convert starch to
glucose
Proteases Washing powder
The exam questions