BPHARM SEM 2 ENZYMES BIOCHEM

1. UNIT: I
ENZYMES
By Mrs. Sonali Nikam
Assistant professor
Faulty of Pharmaceutical Sciences

2. Content
• 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

3. Introduction
• Enzymes are biocatalyst i.e. the catalysts of life.
• Enzymes may be defined as biocatalysts synthesized by
living cells. They are protein in nature (exception - RNA
acting as ribozyme), colloidal and thermolabile in
character, and specific in their action.

4. Classification
ENZYMES
Intracellular
Eg.- Catalase
Extracellular
Eg.-Digestive

5. IUB Classification - OTHLIL
ENZYMES
Oxidoreductases Transferases Hydrolases Lyases lsomerases Ligases
International Union Of Biochemistry

7. Nomenclature
• In the early days, the enzymes were given names by their
discoverers in an arbitrary manner. For example, the names pepsin,
trypsin.
• Trivial names - 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.

8. Properties
• Enzymes have protein in nature.
• Each enzymes has- Tertiary structure and specific
conformation-Essential for catalytic activity.
• Functional unit of enzymes- Haloenzymes which often
made up of apoenzymes (protein part) and coenzymes
(non-protein part).
• Prosthetic group - non-protein moiety tightly (covalently)
binds with the apoenzymes

9. Monomeric
enzyme
• Made up of single polypeptide
• Eg. Ribonuclease, Trypsin
Oligomeric
enzymes
• More than one polypeptide.
• Eg. Lactate Dehydrogenase

10. • Factor affecting on enzyme activity
1. Concentration of enzyme
• Concentration of enzyme directly proportional to Catalytic activity
of enzymes.
2. Concentration of substrate
• Concentration of substrate gradually increases the velocity of
enzyme reaction increase within the limited range of substrate
levels.
• A rectangular hyperbola is obtained.
• Three distinct phases of the reaction are observed in the graph (A-
linear; B-curve; C-almost unchanged).

11. 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 the
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.

12. Enzyme kinetics (Michaelis plot, Line Weaver Burke plot)
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).
Here kl , k2 and k3 represent the velocity constants for the respective
reactions, as indicated by arrows.
Km the Michaelis-Menten constant (or Brig’s and Haldane's constant), is
given by the formula

13. The following equation is obtained after suitable algebraic manipulation.
Where,
V= Measured velocity,
Vmax= Maximum velocity
S= Substrate concentration,
K= Michaelis- Menten constant.
Assume V=1/2 Vmax, then equation
(1) becomes,…………

14. • Km or The Michaelis-Menten constant is defined as the substrate concentration
(expressed in moles/l) to produce half-maximum velocity in an enzyme
catalysed reaction.
• It indicates that half of the enzyme molecules (i.e. 50%) are bound with the
substrate molecules when the substrate concentration equals the Km value.
• Low Km –High affinity between E and S.
• High Km- Low affinity between E and S.
• For majority enzyme Km value in the range 10-5 to 10-2

15. Lineweaver-Burk double
reciprocal plot : For the
determination of K, value, the
substrate saturation curve is
not very accurate since Vmax
is approached asymptotically.
By taking the reciprocals of the
equation (1), a Straight line
graphic representation is
obtained.

16. Enzyme inhibitors with examples
ENZYMES
Inhibitors
Reversible
Competitive
Eg-Succinate
Dehydrogenase
Non-Competitive
Eg-Heavy metal
ions Hg, pb
Irreversible
Eg-lodoacetate to
Papain and Gly 3-
PDH
Allosteric
Eg-Palmitate
Acetyl CoA
Carboxylas
• Enzyme inhibitor is defined as
a substance which binds with
the enzyme and brings about a
decrease in catalytic activity of
that enzyme.
• The inhibitor may be organic
or inorganic in nature.

17. • Reversible Inhibitors
• The inhibitor binds non-covalently with enzyme and the enzyme
inhibition can be reversed if the inhibitor is removed.
Reversible
Competitive
inhibition
Non-competitive
inhibition
Un-competitive
inhibition

18. 1. Competitive inhibition:
 The inhibitor (I) which closely resembles the real substrate (S) is regarded as a
substrate analogue.
 Inhibitor Competes with substrate and binds at the active site of the enzyme but
does not undergo any catalysis.
 The inhibition could be overcome by a high substrate concentration.
 Degree of competitive inhibition determined by relative concentration of S and I
and their affinity with E.
 Km Increase and Vmax-Unchanged.
 Eg- Succinate Dehydrogenase, namely, malonic acid, glutaric acid and oxalic
acid, have structural similarity with succinic acid and compete with the
substrate for binding at the active site of SDH

20. 2. Non-Competitive inhibition:
 The inhibitor binds at a site other than the active site on the enzyme surface.
 This binding impairs the enzyme function. The inhibitor has no structural
resemblance with the substrate.
 The inhibitor does not interfere with the enzyme-substrate binding.
 But the catalysis is prevented, possibly due to a distortion in the enzyme
conformation.
 Km - Unchanged and Vmax lowered.
 Overall relation in non-competitive inhibition
is represented as.
 Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-competitively inhibit the
enzymes by binding with cysteinyl sulfhydryl groups.

21. Km Increase and Vmax-
Unchanged
Km - Unchanged and Vmax
lowered

22.  Irreversible inhibition:
• The inhibitors bind covalently with the enzymes and inactivate them, which is
irreversible.
• These inhibitors are usually toxic poisonous substances.
• Iodoacetate is an irreversible inhibitor of the enzymes like papain and
glyceraldehyde 3-phosphate dehydrogenase oxaloacetate combines with sulfhydryl
(-SH) groups at the active site of these enzymes and makes them inactive.
• Disulfiram (Antabuse @) is a drug used in the treatment of alcoholism. lt
irreversibly inhibits the enzyme aldehyde dehydrogenase.
• Penicillin antibiotics act as irreversible inhibitors of serine

23.  Allosteric inhibition:
• When an inhibitor binds to the enzyme, all the active sites of the protein complex of
the enzyme undergo conformational changes so that the activity of the enzyme
decreases.
• Allosteric inhibitor is a type of molecule which binds to the enzyme specifically at
an allosteric site.
• The enzymes possess additional sites, known as allosteric sites besides the actives
site. Such enzymes are known as allosteric enzymes.
• Allosteric site brings about a conformational change in the active site of the
enzyme, leading to the inhibition
• Eg. Palmitate inhibits to Acetyl CoA carboxylase
• Glucose 6-phosphate to Hexokinase

24. Regulation of enzymes: enzyme induction and repression
Numerous enzymes are responsible for the multiplication and division of
cells in microorganisms. Microbes need certain enzymes to establish an
infection, from a health perspective. Certain enzymes remain active at all
times. Constitutive enzymes are the ones that are active all the time. On
the other hand, other enzymes are only active occasionally, when their
product is needed.
Inducible enzymes are those enzymes in which the activity is controlled
by microorganisms such as bacteria. Survival of the microorganism
depends on this ability. Such enzymes, if constantly active, could over-
produce a compound, resulting in an energy drain for the microbe. As
well as being able to respond quickly to whatever condition they are
geared to, inducible enzymes must also behave predictably.

25. Induction and repression serve the twin ends of controlling activity and
speeding up response.
RNA polymerase, which binds to DNA, is responsible for both induction and
repression of gene expression. In general, the RNA polymerase binds directly
to the DNA sequence that encodes a protein. That region is referred to as the
operator. Positioning the polymerase correctly is essential to advancing the
molecule from one end of the DNA to the other so that information can be
interpreted as it moves along.
RNA polymerase binds to the operator region because of its tri-dimensional
shape, An effector is a molecule that can affect the operation of an operator. A
polymerase effector can alter the shape of the polymerase-binding region,
allowing it to bind more efficiently and easily.
Induction is result of this process. Effectors, on the other hand, can attach to
operators and alter

26. As a result of the presence of a specific molecule, enzymes are induced to be
produced.
A compound that induces an enzyme is called an inducer molecule.
An inducer molecule is combined with a repressor molecule in the induction process.
An inducer can bind to a repressor and block its function, which is to bind to a
specific region known as an operator.
Operators act as catalysts to promote the transcription of genes into messenger RNA
that acts as a template for protein synthesis. RNA polymerase is a molecule that
binds to operators to initiate transcription.
By binding to the repressor, the inducer prevents the repressor from suppressing
transcription, allowing transcription of the gene encoding the inducible enzyme.
The inducing molecule overrides the default mode of transcription, which is
repression. Lactose (lac) operons, which operate based on induction, are very well
characterized in bacteria.

27. Repression of enzymes occurs when repressor molecules inhibit the production
of enzymes.
Feedback inhibition is the typical mechanism of repression.
An amino acid can function as a repressor molecule in a series of enzyme
catalyzed reactions if its end product is that amino acid.
The repressor often combines with another molecule and blocks the operator's
action. It is possible for repressors and polymerase to compete for binding sites on
the operator.
It is also possible for the repressor duo to bind directly to the polymerase to
inhibit the subsequent binding of the operator region by causing the polymerase to
change its shape. No matter what happens, the result is that transcription of that
particular gene is halted.
Genes that are blocked in enzyme repression typically function as receptors for
the first enzyme involved in repressor synthesis.

28. Repression inhibits the synthesis of all enzymes associated with the metabolic
pathway. The bacteria are thus able to conserve energy. It would then be
necessary to produce enzymes, which would involve a large metabolic cost, and
would not play an essential role in cellular processes.
A blockage of an enzyme is usually the first step in a pathway that leads to
repression.
The repression/induction cycle is triggered by nutrient concentration, pH, or other
conditions that affect the enzyme's function. As such, repression interferes with
the production of all enzymes involved in the metabolic process.

29. Allosteric Enzyme Reaction
It is an allosteric enzyme that has a binding site other than the active site for
effector molecules. The binding changes the catalytic properties of the enzyme
because it changes its conformation.
Effector molecules can be inhibitors or activators. A well-regulated biological
system is present in all organisms. Various regulatory mechanisms operate in the
body to monitor and adapt to changes in the inside and outside environment.
Gene expression, cell division, hormone production, metabolism, and enzyme
production are all regulated to ensure proper development and survival.
Enzymes are regulated by allostery, in which binding at one site affects the
Homotropic regulation - Substrate molecules can also act as effectors here. A
major component of this process is the activation of enzymes, also known as
cooperativity, i.e., oxygen binds to hemoglobin.

30. Heterotopic regulation - The substrate is not the same as the effector.
Activators or inhibitors of the enzyme can be used, e.g., CO2 binding to
hemoglobin.
The two types of allosteric regulation are inhibition and activation, based on the
action of the regulator.
1. Allosteric inhibition
If a protein complex of an enzyme is bound to an inhibitor, the enzyme's
activity decreases due to conformational changes at all the active sites.
2. Allosteric activation –
Activators bind to active sites and increase their function, which in turn
increases substrate binding.

31. Therapeutic applications of enzymes and isoenzymes

32. Diagnostic applications of enzymes

33. Coenzymes: Structure and biochemical functions
The non-protein, organic, low 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);
Term prosthetic group is used When a non-
protein moiety is tightly bound to the enzyme
biochemical functions
• Coenzymes are often regarded as
the second substrates or co-
substrates.
• Participate in various reactions
involving transfer of atoms or
groups like hydrogen, aldehyde,
keto, amino, acyl, methyl, carbon
dioxide
• Play a decisive role in enzyme
function
Coenzymes