Dr. B. Victor presented on the biochemical principles of enzyme action. Some key points include: enzymes are proteins that act as catalysts to lower the activation energy of biochemical reactions; they have an active site that binds specifically to substrates; the lock and key and induced fit models describe how enzymes and substrates interact; factors like temperature, pH, and inhibitors can impact an enzyme's activity level; and coenzymes, isoenzymes, and allosteric enzymes are types of modified enzymes. Dr. Victor has over 30 years experience teaching biochemistry and guiding PhD students.

1. BIOCHEMICAL PRINCIPLES OF
ENZYME ACTION
Presented by
Dr. B. Victor., Ph.D.
Email: bonfiliusvictor @gmail. com
Blog: bonvictor.blogspot.com

2. Presentation outline
 Definition and explanation
 Properties, origin, number/cell and size of enzymes.
 All enzymes are proteins-evidences
 Enzyme catalysts and chemical catalysts.
 Classification and naming of enzymes.
 Principle-active site-characteristics.
 Mechanism of enzyme catalysis-models.
 Activation energy and equilibrium constant.
 Enzyme kinetics-Michaelis constant
 Properties of enzyme.
 Coenzymes, isoenzymes, proenzymes and allosteric
enzymes

3. Definition and explanation
Enzymology is the study of enzymes
• Study of understanding the nature and
functions of enzymes.
Catalysts
• Catalysts are chemicals that speed up
the rate of biochemical reactions.

4. Definition of enzymes
Enzymes
• Enzymes are proteins functioning as
catalysts that speed up reactions by
lowering the activation energy.
• The enzyme catalysts regulate the
structure and function of cells and
organisms.

5. Properties of enzymes
Enzymes catalyze Enzymes accelerate Enzymes decrease the
biochemical the velocity of a energy of activation
reactions in living cell. biochemical reaction of substrates.
The chemical nature of
Enzyme does not
an enzyme is not
change the equilibrium
changed by entering
constant of a reaction
a biochemical reaction

6. Origin of enzyme chemistry
In 1850s, Louis Pasteur presented a theory that sugar
is converted into ethanol in yeast by ‘ferments’.
He recognized that ferments was acting as a catalyst
in fermentation
The word ‘enzyme’(en=in; zyme-= yeast) was coined
by German physiologist Wilhelm Kuhne in 1877.
Enzyme literally means ‘in yeast’.
In 1897 Eduard Buchner discovered that
fermentation of the sugar was possible with the
enzyme ’zymase’. He received the Nobel prize for
chemistry in 1907.

7. Number of enzymes per cell
A bacterium like Escherichia coli has about
4228 proteins of which almost 1701 of
them are enzymes.
Mammals have more than 10 times the
number of proteins and enzymes found in
E. coli.
The human body makes approx. 22
digestive enzymes .

8. Size of enzymes
Enzymes are globular proteins
with three dimensional shape.
Size range from 62 amino acid
residues to over 2,500 in
animal fatty acid synthetase.
Molecular mass 12 kd to 1000
kd.

9. “All enzymes are proteins”-
justification
UV absorption at
280nm
Have peptide Amphoteric
bonds nature
Influenced by pH Have isoelectric
and temperature point
nondialysable

10. Difference between enzyme catalysts
and chemical catalysts
enzyme catalysts chemical catalysts
• Protein in nature • Non-protein in nature
• Catalyses a specific reaction • Catalyze different reactions
• Catalysis occur via active site • Catalysis takes part as a
of enzymes. whole.
• The enzyme does not return
• Catalyst always return to its
to their original state after a
biochemical reaction.
original state.
• Generally produced by living • Reacts outside living cells.
cells and acts inside living
cells.

11. Classification of enzymes
Site action- Source of enzyme- Chemical composition –
Intracellular, Plants, animals, Simple, metallo- or
extracellular microorganisms conjugated
Type of reaction – Type of substrate –protein-
protease
Hydrolase,
Carbohydrate –
Oxidase, carbohydrases
dehydrogenase Lipids - lipase

12. 6-major classes of enzymes
Oxidoreductases • Catalyze redox reactions(6-sub classes).
• Transfer functional groups between donors and
Transferases acceptors.
Hydrolases • Catalyze hydrolysis of substrates
Lyases • Catalyze removal of a group other than Hydrolysis.
Isomerases • Catalyze inter-molecular rearrangement
Ligases • Catalyze the union of two molecules.

13. Enzyme nomenclature
Naming the enzymes
Named by
To add ‘suffix’to Retain old groups that
the name of the traditional catalyse similar
substrate To add suffix-
names- chemical Named from the
’lytic’ to denote
Urea-urease reactions species of origin
Ptyalin splitting.
Arginine- Dehydrogenases Papain-papaya
Pepsin Proteolytic
arginase Oxidases Ficin-ficus
Renin lipolytic
Tyrosine- Proteinases
tyrosinase trypsin
lipases

14. Each enzyme is represented by 4
numbers as per enzyme commission
First number
class
indicates
Second
number subclass
indicates
Third number Sub-
indicates subclass
Fourth Serial
number number in
indicates that class
e.g. lactate dehydrogenase (1.1.1.27)

15. Principle of enzyme action
Enzyme-
Enzyme Substrate substrate
(E) (S) Complex
(ES)
Enzyme-
Substrate Enzyme Products
Complex (E) (P)
(ES)

16. Basic principle of enzyme action
Mechanism of enzyme action Details of the mechanism
• The metabolite on which
the enzyme act is the
substrate.
• The substrate when
activated by an enzyme,
combines with the enzyme
to form the enzyme-
substrate complex.
• This in turn dissociates
releasing products and
leaving the enzyme free.

17. Active site • The site of attachment of
of enzyme substrate is active site.
• This site is variously known
as active centre, catalytic site
or substrate site.
• The active site has two
components, the catalytic
and specificity site.
• The active site has two
functions-to bind to a specific
substrate and to catalyze its
the chemical change.

18. Active and regulatory sites of an
enzyme e.g. ribonucleotide reductase

19. Characteristics of • The active site occupies a
active site small portion of enzyme
molecule.
• The active site is well
defined with 3-D shape and
changes its configuration to
bind to a substrate.
• The active site is made up a
few amino acid side chains.
• The active site binds to a
substrate by weak forces.

20. Mechanism of • Emil Fisher proposed lock-
enzyme catalysis:
Lock and Key Model key model in 1898.
• The enzyme-substrate
union depends on a
reciprocal fit between
enzyme and substrate.
• To stay fit, the active site
must have a
complementary shape.
• This matching resembles
the fitting of a key to a lock.

21. Mechanism of • Induced fit model was
enzyme catalysis: proposed by Koshland in 1958.
Induced Fit Model
• The substrate – active sites are
flexible but structurally not
complementary.
• The binding of a substrate to
the active site is believed to
induce a slight alteration in the
shape of active site.
• After the release of products
the active site returns to its
original configuration.

22. Principle of activation energy
• The energy of activation in a
chemical reaction is a measure
of the energy needed for the
conversion of the substrate
molecules to the reactive
state.
• The enzyme catalysts are more
efficient in lowering the
energy of activation.
• For e.g. the decomposition of
H2O2 require 18kcal/mole of
energy without catalyst, but
only 2 kcal/mole in the
presence of enzyme catalase.

23. Equilibrium constant of biochemical
reaction
• Enzymes do not change
the equilibrium
constant of a reaction ,
but only decrease the
time it takes to reach
the equilibrium.

24. Enzyme Kinetics
(The Michaelis-Menten Model)
Enzyme Kinetics is the study of the rate of enzyme catalyzed chemical reactions .

25. Kinetic parameter, km
(The Michaelis-Menten Model)
• Km is the measure of the affinity of an enzyme for its
substrate. It is generally called Michaelis constant.
• Km values of enzymes range from 10-1 to 16-6 M.
• Km is a constant for a particular set of enzymes and
substrate at optimum temperature and pH.
• Km is independent of the enzyme concentration.
• A high km means weak binding between the enzyme
and its substrate and a low km means strong
binding.

26. Kinetic parameter : Vmax
• Vmax shows how well the enzyme catalyzes a
reaction when the concentration of substrate
is high enough so that all enzyme molecules
exist as ES complexes.
• The maximum rate (Vmax) represents the
turnover number of an enzyme.
• This rate constant is also called the molecular
activity of an enzyme.

27. Specificity of enzyme action
Narrow
• Maltase hydrolyzes only maltose
Enzyme • Urease acts on urea
specificity
Broad • Proteases hydrolyze peptide linkages
Enzyme • Exopeptidases hydrolyze terminal of protein chain.
• Endopeptidases hydrolyze within protein chain
specificity
• L-amino acid oxidase acts on L-amino acids.
stereo specificity • D-amino acid oxidase acts on D-amino acids

28. Properties of enzymes
• Enzymes are dynamic proteins.
• Enzyme activity is specific which may be relative or
absolute.
• Most enzymes are soluble in water.
• Enzymes are colloidal in nature.
• Small quantity is required for enzyme action
• Temperature –dependent activity – Vant Hoff’s law
states that the velocity of chemical reaction is at least
doubled by a rise of 100C (Q10).
• pH dependent activity – enzymatic action is greatly
influenced by changes in hydrogen ion concentration.

29. Effect of temperature on
enzyme activity
• Temperature greatly
affect the activity of
enzymes.
• If the temperature is
increased by 100C , the
rate of enzyme action is
doubled.
• Denaturation of enzymes
occur between 40-600C.
• Maximum enzyme
activity occurs at
optimum temperature.

30. Effect of pH on enzyme activity
• Hydrogen ion
concentration also have
an influence on enzyme
activity.
• For most enzymes, the
effective pH range is 4.0-
9.0.
• Beyond these limits,
denaturation of enzymes
take place.
• Optimum pH for pepsin is
2.0 and for trypsin 8.0

31. Apo Holo
coenzyme
enzyme enzyme

32. Prosthetic groups of apoenzyme
• Loosely bound metal ions
• Zn2+ =carbonic anhydrase
cofactors • Cu 2+ = cytochrome oxidase
• Strongly bound organic molecules
Prosthetic • Heme, flavins, biotin
groups
• Strongly bound low molecular weight non-protein.
coenzymes
• TPP, FMN, FAD, NAD, NADP

33. Isoenzymes
• Certain enzymes occur in mult-imolecular forms
in the same organism.
• They are structurally related but catalyze the
same reactions using different kinetic
parameters.
• E.g. Lactate dehydrogenase (LDH)
Alkaline phosphatase
Glutamate oxaloacetate trans aminase
Creatine phosphokinase

34. Zymogens or pro-enzymes:-enzymes synthesized
in a catalytically inactive form.
Zymogens Active enzymes
• Prothrombin • Thrombin
• Chymotrypsinogen • Chymotrypsin
• Trypsinogen • trypsin
• Pepsinogen • Pepsin
• Procarboxypeptidase • Carboxypeptidase
• Prophospholipase • Phospholipase
• Proelastase • Elastase
• Fibrinogen • Fibrin

35. Allosteric enzymes
• Allosteric enzymes possess one or more allosteric
sites (regulatory sites), which are distinct from
the catalytic sites.
• Allosteric sites specifically binds to the inhibitory
modulators.
• Inhibitory modulators may be positive or
negative
• They modulate an enzyme at any substrate
concentration.
• The degree of inhibition is related to the
concentration of the inhibitor.

36. Enzyme inhibitors
Irreversible Competitive
inhibitors inhibitors
Enzyme
inhibitors
reversible Uncompetitive
inhibitors inhibitors
Noncompetitive
inhibitors

37. Meaning of enzyme inhibitors
• Irreversible inhibitors – inhibition of enzyme
activity by combining with active site.
• Competitive inhibitors – inhibition of enzyme
activity by competing with active site.
• Uncompetitive inhibitors - inhibition of enzyme
activity by combining with allosteric site.
• Noncompetitive inhibitors – inhibition by binding
with both to the free enzyme and ES at the
allosteric site.

38. Research objectives in
the study of enzymes
Molecular • Structural aspects :Primary, secondary, tertiary and
quaternary
structure
Protein • PH and temperature stability, isoelectric point
• Electrophoretic mobility, molecular mass, spectroscopic
properties properties.
Catalytic • Specificity, kinetic properties, catalytic mechanism
• Regulatory mechanism, thermodynamic constants
activities
Physiological
• Cellular location, metabolic role, metabolic flux.
function

39. Summary
• Enzymes are dynamic proteins that accelerate
biochemical reactions.
• Each enzyme acts on a specific reactant, the substrate.
• Enzymes are characterized by greater activity,
specificity and susceptibility to the influence of pH,
temperature and other environmental changes.
• Enzymes act in the presence of non-peptide cofactors
or coenzymes.
• An enzyme lacking its cofactor is called apoenzyme and
the active enzyme with its co-factor, the holoenzyme.

40.  Dr.B.Victor is a highly experienced professor, recently
retired from the reputed educational institution- St.
Xavier’s College, Palayamkottai, India-627001.
 He was the dean of sciences, IQAC coordinator and
assistant controller of examinations.
 He has more than 32 years of teaching and research
experience
 He has taught biochemistry at UG and PG levels and
guided 12 Ph.D scholars.
 Send your comments to : bonfiliusvictor@gmail.com