It covers enzyme kinetics, classification of enzymes, catalysis, types of catalysis, nomenclature of enzymes, apoenzymes, cofactors, isoenzymes, holoenzyme, factors affecting the rate of chemical reaction, clinical importance of enzymes. It is useful for the students of life sciences and biochemistry as well. The slides help even the teachers teaching basics of enzyme kinetics at the UG and PG levels.

1. VIJAYANAGARA SRI KRISHNADEVARAYA UNIVERSITY, BALLARI
ENZYMES
Dr. Nagabhushan CM
Assistant Professor, DOZ,
VSK University, Ballari

2. Contents:
Meaning
General principles
Terminology
Catalysts
Types of catalysts
Nomenclature
Classification
Thermodynamics
Enzyme kinetics
Regulation of enzyme activity
Active site
Cofactors
Holoenzymes
Factors affecting enzyme catalysis
Inhibitors
Diagnostic significance of Isoenzymes
References

3. ENZYMES
ARE THE PROTEINS WITH CATALYTIC PROPERTIES

4. ENZYMES
• proteins that play functional biological roles
• responsible for the catalysis of all chemical
reactions that take place in living organisms
– acceleration of reactions by factors of 106 to 1017
• biological catalysts that bind and catalyse the
transformation of substrates.
• many enzymes have the three-dimensional structures.

5. General principles
• A catalyst accelerates a reaction without being consumed
• the rate of catalysis is given by the turnover number
• a reaction may alternatively be “promoted” (accelerated,
rather than catalysed) by an additive that is consumed
• a heterogeneous catalyst is not dissolved in solution;
catalysis typically takes place on its surface.
• a homogeneous catalyst is dissolved in solution, where
catalysis takes place.
• all catalysis is due to a decrease in the activation barrier.

6. General principles
• their presence DOES NOT AFFECT the nature and properties
of the end products.
• They are highly specific in their action that is each enzyme can
catalyse one kind of substrate.
• SMALL AMOUNT of enzymes can accelerate chemical
reactions.
• Enzymes are SENSITIVE to change in pH, temperature and
substrate concentration.

7. ENZYME TERMINOLOGY
• SUBSTRATE: A REACTANT WHICH BINDS TO ENZYME
• PRODUCT: THE END RESULT OF A REACTION
• ACTIVE SITE: ENZYME’S CATALYTIC SITE WHERE THE
SUBSTRATE FITS INTO.

8. AS CATALYSTS
• Efficient at LOW CONCENTRATIONS.
• Not consumed during the reaction.
- Transformation of 20 to 36 x 106 substrates / min.
• Do not affect the equillibrium of reversible chemical reactions.
• Most enzymes operate at extreme reaction conditions.
• Catalysis accelerates the reaction.
- Free energy of the reaction remains unchanged
- Equillibrium constant remains unchanged.

9. AS CATALYSTS
• Enzyme controlled reactions proceed 108 to 1011 times faster than
corresponding non-enzymatic reactions

10. Types of catalysts
APPROXIMATION
the catalyst brings the reactants together, increasing their effective
concentrations and orients them.
ELECTROSTATIC CATALYSIS
stabilization of charges developed during chemical reaction.
COVALENT CATALYSIS
catalyst forms a covalent intermediate that reacts faster than
uncatalysed reaction.
STRAIN & DISTORTION
the approach of substrate serves to provoke a conformational change in
the enzyme.
Strain and distortion in the substrate are essential for the catalysis.

11. NOMENCLATURE
Enzymes are named based on THE NAME OF THE SUBSTRATE IT
CATALYSES and SUFFIX IN to the old trivial names to PEPSIN, RENIN.
ex:
substrate enzymes Products
LACTOSE LACTASE GLUCOSE AND
GALACTOSE
MALTOSE MALTASE GLUCOSE
CELLULOSE CELLULASE GLUCOSE
LIPID LIPASE GLYCEROL AND
FATTY ACID
STARCH AMYLASE MALTOSE
PROTEIN PROTEASE PEPTIDES AND
POLYPEPTIDES
NUCLEIC ACID NUCLEASE NUCLEOBASES

12. NOMENCLATURE
Each enzyme has a NUMERICAL CODE formed of FOUR DIGITS
separated by dots:
1. First digit denotes the CLASS of enzyme.
2. Second digit denotes the FUNCTIONAL GROUP.
3. Third digit denotes the CO-ENZYME.
4. Fourth digit denotes the SUBSTRATE.
For example 1.1.1.1 enzyme, where
1 means oxido-reductase, 1.1 means that the functional group is
hydroxyl group (-OH), 1.1.1 means NAD is the coenzyme and
1.1.1.1 means alcohol.
So, 1.1.1.1 means alcohol dehydrogenase enzyme.

13. CLASSIFICATION OF ENZYMES
Developed by INTERNATIONAL ENZYME COMMISSION.
Based on TYPE OF REACTION CATALYSED.
Each class is further subdivided into further divisions.

14. CLASSIFICATION OF ENZYMES
class Reaction type examples
oxidoreductase Redox Lactate dehydrogenase
transferases Move Chemical Group Hexokinase
hydrolases Hydrolysis: cleaving bonds with
transfer of functional groups.
Lysozyme
lysase Non-hydrolytic bond cleavage Fumerase
isomerase Intramolecular group transfer Triose phosphate isomerase
ligases Synthesis of new covalent bonds
between substrates
RNA polymerase

15. THERMODYNAMICS
• All chemical reactions have energy barriers between reactants and products.
• The difference in transitional state and substrate is called activational barrier.
• Only a few substances cross the activation barrier and change into products.
• That is why rate of uncatalyzed reactions is much slow.
• Enzymes provide an alternate pathway for conversion of substrate to products.
• Enzymes accelerate reaction rates by forming transitional state having low
activational energy.
• Hence, the reaction rate is increased many folds in the presence of enzymes.
• The total energy of the system remains the same and equilibrium state is not
disturbed.

16. KINETICS
Kinetic analysis reveals the number and order of the individual steps by which
enzymes transform substrate into products
Studying an enzyme's kinetics can reveal the catalytic mechanism of the
enzyme, its role in metabolism, how its activity is controlled and being
inhibited.

17. KINETICS
Activation Energy (Ea):
“The least amount of energy needed for a chemical reaction to take
place.”
Enzyme (as a catalyst) acts on substrate in such a way that they lower the
activation energy by changing the route of the reaction.
The reduction of activation energy (Ea) increases the amount of reactant
molecules that achieve a sufficient level of energy, so that they reach the
activation energy and form the product.

18. REGULATION OF ENZYME ACTIVITY
LOCK AND KEY MODEL proposed by EMIL FISCHER in 1894
INDUCED FIT MODEL proposed by DANIEL KOSHLAND in 1958

19. REGULATION OF ENZYME ACTIVITY
LOCK AND KEY MODEL proposed by EMIL FISCHER in 1894
Enzyme may be
used again
Enzyme-
substrate
complex
E
S
E
E
P
P
Reaction
E
© 2017 Paul Billiet ODWS

20. REGULATION OF ENZYME ACTIVITY
INDUCED FIT MODEL proposed by DANIEL KOSHLAND in 1958
(a) Free Hexokinase (b) with glucose substrate

21. ACTIVE SITE
The active site of an enzyme is the region that binds substrates,
co-factors and prosthetic groups and contains residue that helps to
hold the substrate.
Active sites generally occupy less than 5% of the total surface
area of enzyme.
Active site has a specific shape due to tertiary structure of protein.
A change in the shape of protein affects the shape of active site
and function of the enzyme.

22. ACTIVE SITE
The active site of an ENZYME has
BINDING SITE: IT CHOSES THE SUBSTRATE AND BINDS IT
TO THE ACTIVE SITE
CATALYTIC SITE: IT PERFORMS THE CATALYTIC ACTION
OF ENZYME.

23. COFACTORS
• An additional non-protein molecule needed
by some enzymes to help the reaction.
• Tightly bound cofactors are called
prosthetic groups (inorganic metal ions)
• Cofactors that are bound and released easily
are called coenzymes derived from Vit-B
(FAD, NAD etc).
• Many vitamins are coenzymes.
Nitrogenase enzyme with
iron,molybdenum and ADP
as cofactors
© 2017 Paul Billiet ODWS

24. HOLO ENZYMES
• DNA polymerase is a HOLOENZYME that catalyses the
polymerization of deoxy-ribonucleotide into a DNA strand,
USES Mg ions for catalytic activity.
• Liver dehydrogenase USES Zn ions for its activation.

25. FACTORS AFFECTING RATE OF ENZYME ACTION
1. Enzyme concentration.
2. Substrate concentration (Michaelis constant = Km)
3. pH (1.5for Pepsin/ 7.5 for steapsin/ 6.8 for ptyalin)
4. Temperature (37-40 oC), denature after this limit.
5. Concentration of coenzymes.
6. Concentration of ion activators (Cl activates
ptyalin / Ca – thrombokinase)
7. Time
8. Inhibitors

26. FACTORS AFFECTING ENZYMES
1. pH
2. Temperature
3. Substrate concentration
4. Inhibitors

27. FACTORS AFFECTING ENZYMES
pH, Temperature, Substrate concentration, Inhibitors
Enzyme
activity
Trypsin
Pepsin
pH
1 3 5 7 9 11
© 2017 Paul Billiet ODWS
Optimum pH values

28. FACTORS AFFECTING ENZYMES
pH, Temperature, Substrate concentration, Inhibitors
© 2017 Paul Billiet ODWS Temperature / °C
Enzyme
activity
0 10 20 30 40 50
Increasing
number of
collisions (Q10)
Denaturation
Optimum

29. FACTORS AFFECTING ENZYMES
pH, Temperature, Substrate concentration, Inhibitors
© 2017 Paul Billiet ODWS
Reaction
velocity
Substrate concentration
Reaction
velocity
Substrate concentration
Vmax
NON ENZYMATIC REACTION ENZYMATIC REACTION

30. FACTORS AFFECTING ENZYMES
pH, Temperature, Substrate concentration, Inhibitors
A SUBSTANCE THAT DIMINISHES THE VELOCITY OF THE ENZYME
CATALYSED REACTION IS CALLED AN INHIBITOR.

31. INHIBITORS
1. Competitive inhibitor: the inhibitor competes with the substrate for the
active site. E S complex is reduced and E I complex is formed.
2. Uncompetitive inbibitor: inhibitor instead binds to ANOTHER SITE
known as ALLOSTERIC SITE. ex: drugs that treat methanol poisoning.
Tetramethylene sulfoxide of liver alcoholdehydrogenase.
3. Mixed inhibition: both E S and E I complexes are formed.
4. Non-competitive inhibition: it is a special case where Inhibitor has the
equal affinity for either ENZYME OR E S COMPLEX.
5. Irreversible inhibition: covalent attachment of inhibitor to the enzyme.
Ex: aspirin
6. Suicide inhibition: enzyme’s active site is converted into REACTIVE
FORM.

32. DIAGNOSTIC SIGNIFICANCE OF ISOENZYMES
CLINICALLY IMPORTANT ENZYMES
ENZYME CONCENTRATION HIGH CONCENTRATION IN DIAGNOSIS
Lactate dehydrogenase (LDH) 60 IU/L MI, Leukemia, Acute Hepatitis
Transaminase (aspartyl
transaminase=AST)
MI
Alalnine transaminase (ALT) LIVER DISORDER
Acid phosphatase Prostrate carcinoma
Isocitrate dehydrogenase Brain tumour / meningitis
Gamma glutamyl transpeptidase (g-GT) Liver damage indicator of alcoholism
Isoenzyme LDH 1 heat resistant MI, Kidney disease
Isoenzyme LDH 4 heat labile Lung /spleen /pulmonary infarction

33. Acknowledgement and references
• Woodbury: Biochemistry for the Pharmaceutical Sciences
• Lehninger: Principles of biochemistry
• Lippincott: Biochemistry
• Harper's Illustrated Biochemistry
• Mushtaq Ahmed: Essentials of Medical Biochemistry
• Pfeiffer, J: Enzymes, the Physics and Chemistry of Life
• Martinek, R: Practical Clinical Enzymology.
• Kalavani satish, assistant professor, PIMS, Panipath
• Enzymes: Dr. Salva Abo El-khair
• open resource site from google.

34. THANK YOU
Dr. Nagabhushan CM, Assistant Professor,
Dept. of studies in Zoology, VSK University, Ballari