Biochemical aspects of enzyme, classification, mechanism of action, enzyme inhibition, importance of enzyme for mbbs course

1. Enzyme
Dr. Farhana Atia
Assistant Professor
Department of Biochemistry
Nilphamari Medical College, Nilphamari
Email: farhana.atia@gmail.com

2. ENZYMES
Enzymes are
– colloidal,
– heat labile,
– organic catalyst (biocatalyst),
– protein in nature (except rybozyme),
– Non dialyzable
– Catalyze biochemical reaction.
Substrate: Bind & react with enzyme at its
active site
Product: Fate of substrate

3. Enzyme has two site-
• Active site: Substrate binds here
• Allosteric site : Another site which can alter the
enzyme action. Inhibitor/ stimulator binds here

4. IUB Classification
Class Reaction catalyzed
Oxidoreductases oxidation-reduction
Transferases transfer of moieties such as glycosyl,
methyl, or phosphoryl groups
Hydrolases hydrolytic cleavage of C-C, C-O, C-N bond
Lyases cleavage of C-C, C-O, C-N bond by atom
elimination, generating double bond
Isomerases geometric / structural changes within a
molecule
Ligases/
synthetase
joining together of two molecules coupled
to the hydrolysis of ATP

5. According to location
1. Plasma specific enzyme/ Functional enzyme
– Present in plasma & act continuously. E.g.
Lipoprotein lipase, Plasminogen
2. Nonfunctional enzyme
1. Secretory enzyme: Digestive enzyme
2. Intracellular enzyme
• In plasma: Present in very small amount & not
perform any function
• Level increased in blood after tissue damage
• AST, ALT

6. Many enzyme contain small non protein molecule & metal
ions that participate directly in substrate binding / catalysis,
termed prosthetic group, cofactor, coenzyme
Apoenzyme
Coenzyme
Prosthetic
group
Metal ion
Cofactor/
Non protein
part
Holoenzyme
Protein
part

7. Coenzyme: Heat stable, low molecular weight, non protein,
organic compound required for enzyme activity, usually
derived from vit B complex
Vitamin Coenzyme
B1-Thiamine Thiamine pyrophosphate (TPP)
B2-Riboflavin Flavin adenine dinucleotide (FAD)
Flavin adenine mononucleotide (FMN)
B3-Niacin Nicotinamide adenine dinucleotide (NAD)
NADP
Biotin Enzyme bound Biotin
B5-Pantothenic acid Coenzyme A
B6-Piridoxine Pyridoxal phosphate (PP)
B12-Cobalamin Methylcobalamin
Deoxyadenosylcobalamin
Folic acid THF (Tetrahydrofolic acid)

8. Co-factor
Organic
(Coenzyme)
Tightly bound
Prosthetic group
Loosely bound
2nd substrate
Inorganic
(Metal ion)
Tightly bound
Metalloenzyme
Loosely bound
Ion activator
Zn, Fe, Cu, Mn,
Mg
2/3rd of enzyme require
metal ion as a co-factor

9. Isoenzyme/ Isozyme
• Physically distinct variant of
same enzyme which catalyze
the same reaction
• Common clinically important
enzyme
– LDH: Lactate dehydrogenase
– CPK/ CK: Creatinine
phosphokinase
– ALP: Alkaline phosphatase

10. Isoenzyme Subunit High level found in
LDH 1 HHHH Myocardium, RBC
LDH 2 HHHM Myocardium, RBC
LDH 3 HHMM Brain, Kidney
LDH 4 HMMM Skeletal muscle
LDH 5 MMMM Liver, Skeletal muscle
CK 1 BB Brain
CK 2 MB Myocardium
CK 3 MM Skeletal muscle
ALP Bone, Liver, Placenta

11. Factors affecting enzyme activity
In laboratory In vivo/ biological system
1. pH 1. Substrate concentration
2. Temperature 2. Allosteric effector
3. Substrate concentration 3. Covalent modification
4. Enzyme concentration 4.Induction & repression of
enzyme synthesis
5. Time & Product
concentration

12. pH
• Optimum pH (5-9), at which
enzyme activity is maximum
• Exception
– Alkaline phosphatase >9
– Pepsin : 1-3
Temperature
• The reaction rate increases with
temperature to a maximum level,
then abruptly declines
• Optimal temperature :40- 50 °C
• Denatured above 60oC

13. Substrate concentration
• Increase velocity of enzyme
as the substrate
concentration increase up to
enzyme saturation
Enzyme concentration
• Directly proportional to the
rate of reaction (provided
sufficient substrate is
present)
Time & product concentration
• Initially linear, than plateau

14. Allosteric
effectors
 Substrate that non
covalently bind with
enzyme other than
the active site
May be-
• Positive effector: ↑
enzyme activity
• Negative effector: ↓
enzyme activity
• Homotrophic effector:
substrate itself act as
effector. Usually +ve effector.
• Heterotrophic effector:
Different from substrate.
Usually negative effector.

15. Covalent modification:
• Many enzyme may be
regulated by covalent
modification by addition or
removal of phosphate group
• Most enzymes are activated
& are some inactivated by
phosphorylation
• Glycogen synthase + PO₄
↓ enzyme activity
• Glycogen phosphorylase +
PO₄ ↑ enzyme activity
Induction & repression
of enzyme synthesis:
 Insulin
• Induce all enzyme of
glycolysis
• Repress enzyme of
gluconeogenesis

16. • The Michaelis-Menten equation is a quantitative
description of the relationship among the rate of
an enzyme- catalyzed reaction [v1], the
concentration of substrate [S] and two constants,
Vmax and km (which are set by the particular
equation).
• The symbols used in the Michaelis-Menten
equation-
– reaction rate [v1]
– maximum reaction rate (V max)
– substrate concentration [S]
– Michaelis-Menten constant (Km)
Michaelis-Menten Kinetics

17. Michaelis-Menten
equation
• Equation states that when [S] equals Km, the initial
velocity is half-maximal.
• Equation also reveals that Km is a constant and may be
determined experimentally from—the substrate
concentration at which the initial velocity is half-maximal

18. • The Michaelis constant Km is the substrate
concentration at which vi is half the maximal
velocity (Vmax/2) attainable at a particular
concentration of enzyme
• It is specific and constant for a given enzyme under
defined conditions of time , temperature and p H
• Km determines the affinity of an enzyme for its
substrate, lesser the Km higher the affinity and vice
versa
• Km value helps in determining the true substrate
for the enzyme.
Km and its significance

19. Enzyme inhibition
• Inhibitor: Any substrate that can inhibit or diminish
the velocity of an enzyme catalyzed reaction.
Enzyme
inhibition
Reversible
Competitive
Non-
competitive
Irreversible

20. Reversible inhibitors:
• Bind with an enzyme by non-covalent bond
Competitive inhibitors:
• Structurally similar to substrate (structural analog)
• Occupies active site
• Compete with substrate for active site
• Action can be reversed by ↑ [substrate]
• Vmax same, Km ↑
• In TCA cycle succinate (S) & malonate (I) compete
for succinate dehydrogenase
• NSAIDs used as competitive inhibitor of
cycloxygenase & prevent PG synthesis

21. Non-competitive inhibition:
• Bind enzymes at sites distinct from the
substrate-binding site
• No structural resemblance to the substrate
• Binding of the inhibitor does not affect binding
of substrate
• Formation of both EI and EIS complexes is
possible
• Vmax ↓, Km same
• Cyanide inhibits cytochrome oxidase
• Fluoride inhibits Enolase and hence glycolysis

23. Irreversible inhibition
• Binds with active site of
enzyme by covalent bond
• Permanently inactivate
enzyme
• Vmax ↓, Km same
• OPC (organo
phosphorous compound)
irreversibly inhibit acetyl
cholinesterase

24. Enzyme specificity
• Act on particular bond or group in closely
related substrate
• Proteolytic enzyme act on peptide bond
Relative/
group
specificity
• Acts on specific substrate
• Urease acts on urea
• Carbonic anhydrase on H₂CO₃
Absolute
specificity
• Acts on only one isomer
• Glucose oxidase acts on β-D-anomer of glucose
• Glucokinase acts on D-glucose
Optical/
stereo
specificity

25. Clinical importance of enzyme
Maintain normal physiology
Diagnosis of disease
Prognosis of disease
Treatment of disease by administration of enzyme or
inhibiting enzyme
Laboratory use

26. Maintain normal physiology
• Total internal environment of human body is
maintained by enzyme otherwise severe disaster
occur
– Na⁺K⁺ ATPase activity
• Without enzyme food can not be digested, absorbed
& metabolized and maldigestion, malabsorption
occur
• Enzyme deficiency can cause disease
– Inborn error of metabolism (PKU- phenyl ketonuria)
– Glycogen storage disease
– G6PD- glucose 6 phosphatase deficiency hemolytic
anemia

27. Diagnosis of disease
• Use of enzyme as diagnostic tool
– Non-functional enzyme released after specific tissue injury
which indicate disease of that organ/ tissue
– Acid phosphatase ↑in Carcinoma prostate
– ALT ↑ in hepatitis
• Isoenzyme determination is very important in case of
MI
– CK₂ ↑ sharply 4-8 hrs after pain & becomes normal within
48-72 hrs
– LDH₁, LDH₂ ↑ 48 hours after infarction & persist 7-10 days
• ELISA employ as a disease indicator (ELISA- enzyme
linked immuno-sorbent assay)

28. Prognosis of disease
• By serial assessment of we can evaluate
prognosis of disease
• Serum ALT for viral hepatitis
Laboratory use
• Clinical analyzer use enzyme as reagent
• Glucose oxidase & peroxidase – used in
estimation of blood glucose

29. Therapeutic use
Therapeutic agent-
• Streptokinase for clot lyses in MI
• Aspirin inhibit cycloxygenase in infection
• Allopurinol inhibit xanthine oxidase in
treatment of gout

30. Thank You