This presentation provides a comprehensive coverage of almost every aspect concerned with enzymes.

1. ENZYMES
Rachel Jacob
S2 M.Sc Zoology
ZOO-15-05-10

2. Enzyme Activity
Enzyme Receptors
Regulation of Enzyme Activity

3. Enzymes are complex proteins of
high molecular weight which
catalyze specific biochemical
reactions in organisms.

4. A peep into the past

5. • At the end of the nineteenth
century, scientist debated on
whether the process of
ethanol formation required
the presence of intact yeast
cells.
• Justus von Liebig (an organic
chemist) argued that the
reaction of fermentation that
produced alcohol, were no
different in vivo and in vitro.

6. • Louis Pasteur(the
renowned biologist)
argued that the process
of fermentation can
occur only within the
confines of an intact
living cell.

7. • Hans Buchner and
Eduard Buchner
prepared yeast extract
which they tried to
preserve by adding
sugar, found that the
extract produced gas
from sugar and it
bubbled continuously
for days.

8. • They discovered that fermentation produced
ethanol and bubbles of carbon dioxide.
• Buchner had shown that fermentation did not
require the presence of intact cells.
• Later it was found that fermentation differed
from chemical reactions carried out in vitro, in
the presence of biological catalysts called
Enzymes.

9. The first evidence that
enzymes are proteins was
obtained in 1926 by James
Sumner of Cornell University
when he crystallized the
enzyme Urease from Jack
Beans.

10. Later RNA catalysts
were also
discovered. They
are called
ribozymes. But the
term enzyme is used
to refer to those
catalysts which are
proteins.

11. Hammerhead Ribozyme

12. They are globular proteins required in small amounts.
They are not altered irreversibly during the reaction.
They have no effect on thermo dynamics of reactions

13. They are very efficient
They are highly specific and regulated
and reversible.
Enzymes increase velocity of reactions
108 to 1013 fold at milder temperature
and pH.

15. Inorganic cofactors are enzyme activators (e.g.,
Mg2+, Ca2+, Co2+)
Organic enzyme cofactors are called
coenzymes.
(e.g., NAD or Coenzyme 1).
Prosthetic groups are cofactors tightly bound to
an enzyme on a permanent basis. (Eg., Haem)

16. Ribozymes are RNA molecules with catalytic
properties. Eg., hammerhead ribozyme, the VS
ribozyme, Leadzyme and the hairpin ribozyme.
Abzymes are antibodies which express catalytic
activity.
A single molecule of an antibody-enzyme, or abzyme,
is capable of catalyzing the destruction of thousands
of target molecules.

20. Initial contact between the active site of an enzyme and
a potential substrate molecule depends on their
collision.
Substrate binding usually involves hydrogen bonds or
ionic bonds Substrate binding is readily reversible.

21. Diagrammatic representation of the active site of the enzyme ribulose bisphosphate
carboxylase oxygenase showing the various sites of interaction between the bound
substrates (RUBP and CO2) and certain amino acid side chains of the enzyme.

22. The lock-and-key model, first suggested in 1894 by the
German biochemist Emil Fischer, explained enzyme specificity
but not the catalytic event.

23. Tight spatial relationship between a glutamic acid (yellow) and a histidine
(green) of the enzyme triosephosphate isomerase respectively and the substrate
(red).

24. The induced-fit model was first proposed in 1958 by
Daniel Koshland.
Substrate binding at the active site distorts enzyme
and the substrate, stabilizing the substrates in their
transition state.
Critical amino acid side chains are brought into the
active site even if they are not nearby in the absence of
substrate.

27. Enzymes lower the etropy of the system
Substrates brought very close together in
precisely the correct orientation to undergo
reaction.

28. Enzymes side chains may be polar or non-polar which
influence distribution of electrons in substrate.

29. • As conformational
changes occur in the
substrate, mechanical
work is performed,
exerting a physical
force on certain bonds
within a substrate
molecule.

30.  The sequence of events at the active site is illustrated using the
enzyme sucrase as an example.
 Sucrase (also known as invertase or b-fructofuranosidase)
hydrolyzes the disaccharide sucrose into glucose and fructose.
 The initial random collision of a substrate molecule—sucrose,
in this case—with the active site results in its binding to
amino acid residues that are strategically positioned there.
 Substrate binding induces a change in the enzyme
conformation that tightens the fit between the substrate
molecule and the active site and lowers the free energy of the
transition state.

32.  This facilitates the conversion of substrate into
products—glucose and fructose, in this case.
 The products are then released from the active site,
enabling the enzyme molecule to return to its
original conformation, with the active site now
available for another molecule of substrate.
 This entire sequence of events takes place in a
sufficiently short time to allow hundreds or even
thousands of such reactions to occur per second at
the active site of a single enzyme molecule.

33. Environmental Conditions
Cofactors and Coenzymes
Enzyme inhibitors

34. Temperature
pH
Substrate concentration

35. • Q10 (the temperature coefficient) = the increase in
reaction rate with a 10°C rise in temperature.
• For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every 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.

36. Temperature / °C
Enzyme
activity
0 10 20 30 40 50
Q10 Denaturation

37. • For most enzymes the optimum temperature is
about 30°C.
• Many are a lot lower,
cold water fish will die at 30°C because their
enzymes denature.
• A few bacteria have enzymes that can withstand
very high temperatures up to 100°C.
• Most enzymes however are fully denatured at
70°C.

38. • 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.

39. Enzyme
activity Trypsin
Pepsin
pH
1 3 5 7 9 11

40. For non-enzymatic reactions
Reaction
velocity
Substrate concentration
The increase in velocity is proportional to the substrate concentration.

41. Reaction
velocity
Substrate concentration
Vmax
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.

43. Inhibition of enzyme activity is important for several
reasons.
o It plays a vital role as a control mechanism in cells.
o Enzyme inhibition is also important in the action of drugs
and poisons.
o Inhibitors are useful as tools in studies of reaction
mechanisms and for treatment of diseases.
Especially important inhibitors are substrate analogues and
transition state analogues.

44. Mechanism of action of substrate analogues

45.  Substrate analogs are used
against infectious disease.
 For example, sulfa drugs
resemble the folic acid
precursor, PABA.

46. • Azidothymidine (AZT) resembles the
deoxythymidine molecule used by HIV to synthesize
DNA using viral reverse transcriptase.
• After binding to the active site, AZT forms a “dead-
end” molecule of DNA that cannot be elongated.

48. IRREVERSIBLE INHIBITORS
• An irreversible inhibitor binds covalently, causing
permanent loss of catalytic activity and are toxic.
• Ions of heavy metals are often irreversible inhibitors,
as are some pesticides and nerve gas poisons.
• These can bind irreversibly to enzymes such as
acetylcholinesterase.
• Inhibition leads to rapid paralysis of vital functions and
therefore to death. E.g., diisopropylflourophosphate.

49. • Diisopropyl fluorophosphate rends the enzyme
molecule permanently inactive.
• Some irreversible inhibitors of enzymes can be used
as therapeutic agents.
• For example, aspirin binds irreversibly to the enzyme
cyclooxygenase-1 (COX-1), which produces
prostaglandins.
• Thus, aspirin is used in low doses as a
cardiovascular protectant.

50. The antibiotic
penicillin is an
irreversible
inhibitor of the
enzyme needed for
bacterial cell wall
synthesis.

51. Reversible Inhibitor
 A reversible inhibitor binds to an enzyme in a noncovalent,
dissociable manner, such that the free and bound forms of the
inhibitor exist in equilibrium with each other.
 The two most common forms of reversible inhibitors are called
competitive inhibitors and noncompetitive inhibitors.
 A competitive inhibitor binds to the active site of the enzyme
and therefore competes directly with substrate molecules for
the same site on the enzyme.

52. Competitive Inhibition
• Competitive inhibitors are reversible inhibitors that
compete with a substrate for access to the active site of
an enzyme.
• Competitive inhibitors must resemble the substrate to
compete for the same binding site, but differ in a way
that prevents them from being transformed into
product.
• Angiotensin converting enzyme (ACE) is a proteolytic
enzyme that acts on a 10-residue peptide (angiotensin I)
to produce an 8-residue peptide (angiotensin II).

53. • Elevated levels of angiotensin II
are a major risk factor in the
development of high blood
pressure (hypertension).
• In the 1960s John Vane and his
colleagues at the Eli Lilly
Company began a search for
compounds that could inhibit
ACE.
• Brazilian pit viper contains
inhibitors of proteolytic
enzymes, and it was found that
one of the components of this
venom, a peptide called
teprotide was a potent
competitive inhibitor of ACE.

54. • It was not a very useful drug
because it had a peptide
structure and thus was rapidly
degraded if taken orally.
• Subsequent efforts to develop
nonpeptide inhibitors of the
enzyme led researchers to
synthesize a compound called
captopril, which became the
first useful antihypertensive
drug that acted by binding to
ACE.

56. Given a sufficient substrate concentration, it
remains theoretically possible to achieve the
enzyme’s maximal velocity even in the
presence of the competitive inhibitor.
• Hence maximal reaction velocity can be
achieved (Vmax remains unchanged)
• But the enyme’s affinity to the substrate is
reduced ( Km increases)

57. Non- competitive inhibition
• In noncompetitive inhibition, the substrate and
inhibitor do not compete for the same binding
site; generally, the inhibitor acts at a site other
than the enzyme’s active site.
• The level of inhibition depends only on the
concentration of the inhibitor, and
increasing the concentration of the substrate
cannot overcome it.

59. Since, in the presence of a noncompetitive inhibitor, a
certain fraction of the enzyme molecules are
necessarily inactive at a given instant, the maximal
velocity of the population of enzyme molecules cannot
be reached.
• Tipranavir is a potent noncompetitive inhibitor of the
protease produced by HIV when it infects a white
blood cell.
• Unlike other inhibitors of this enzyme, such as
ritonavir, tipranavir does not resemble the peptide
substrate of the enzyme, nor does it compete with the
substrate.

62. Considerable progress has been made in the
field of computer-aided drug design.
Scientists can now design a number of
hypothetical inhibitors and test their
binding using complex computer models.

64. • Chemical kinetics studies the rate of chemical
reactions and the factors influencing them.
• Similarly, enzyme kinetics studies the rate of
enzyme catalyzed reactions and the factors
influencing them.

65. Canadian physician Maud Leonara Menten and German
biochemist Leonor Michaelis and (1913) proposed a general
theory to explain enzyme action.
Maud Menten and Leonor Michaelis

67. They tried to explain the relationship between
reaction rate and substrate concentration.

68. Assumptions
They assumed a steady state to pre-exist.
The reaction is in a “steady state” when the concentration of
the enzyme substrate complex remains steady.
The MM equation concerns the initial velocity of the reaction
(v0) when the rateof the backward reaction is negligible.

69. Wherein
v0 = initial reaction velocity
[S] = initial substrate concentration
Vmax = Maximum velocity
Km = substrate conc. at ½ Vmax

70. Plots of the initial velocity at different concentrations of the
enzyme as a function of substrate concentration.
The maximal velocity Vmax is quadrupled once the enzyme
concentration in quadrupled. The Km remains unaltered.

71. Plots of the initial velocity versus substrate concentration for a Substrate S
for which the enzyme has higher affinity and a substrate S’ for which the
enzyme has lower affinity.
Vmax remains same for the same enzyme, but higher the Km, lower the
affinity

72. • Km is a measure of affinity of the enzyme for
its substrate. Greater the Km, lesser is the
affinity.
• Rule of thumb :Intracellular concentration of a
substrate is approximately the same as, or
approximately greater than the Km value of
the enzyme to which it binds.

73. Can you crack this ?
From CSIR Dec 2014

74. And the right answer is..
Option A
Did you get it right?
If so, Congrats!
If not, don’t worry.
A little practice will set you in the right track .

75. Case 1
At low substrate
concentration:
v0= Vmax [S]
Km

76. Case 2
• At intermediate substrate
concentrations, [S]= Km
V0=Vmax/2

77. Case 3
At high substrate
concentrations:
V0=Vmax

78. Lineweaver-Burk Plot
• MM equation yields a hyperbola.
• Accurate prediction of Km and
Vmax values from this hyperbola
requires a large number of data
points.

79. • The Lineweaver Burk plot or the double reciprocal plot helps
overcome this.
• It is simply the reciprocal of MM equation.
• It enables accurate prediction of Km and Vmax from a few data
points and extrapolation becomes easier.

80. Effect of inhibitors on kinetics

83. Enzyme linked receptors form a major type of cell-surface
receptors.
Enzyme-linked receptor proteins either possess an in-built
enzyme or associate with separate enzymes in the cytoplasm.

84. These enzymes are activated upon ligand binding.
They relay the extracellular signal to the nucleus by a
sequence of interactions.
This turns on specific transcription factors, altering gene
expression in the cell.

86. All enzyme-linked receptors share a few common features;
1. Ligand-binding domain
• Extracellular to allow easy access for ligands.
• Strong affinity for specific ligands - allows different ligands that
bind to the same receptor to evoke particular cellular responses.
2. Transmembrane domain
• Contains a series of hydrophobic amino acids.
• Tethers the receptor to the cell membrane.
3. Cytosolic "active" enzyme domain
• Either intrinsic to the receptor or tightly bound via the cytosolic
domain.
• The majority are kinases; they phosphorylate specific threonine,
serine, and tyrosine amino acid residues (THR,S,TY = THIRSTY).

87. Enzyme-linked receptor classes
There are three main types of enzyme-linked receptors:
• 1. Receptor serine-threonine kinases e.g.
transforming growth factor-beta (TGFB) receptors.
• 2. Receptor tyrosine kinases (RTKs) e.g. growth
factor receptors.
• 3. Tyrosine-kinase-associated receptors e.g. cytokine
receptors.

88. Receptor serine/threonine kinases
• There are two units of serine/threonine kinase receptors, both of
which contain an intracellular kinase domain. They are each dimeric
proteins, so an active receptor complex is made up of four units.
• 1. Type I receptors
– Inactive unless in complex with type II receptors.
– Do not interact with ligand dimers.
– Contain conserved sequences of serine and threonine residues near to
their kinase domains.
• 2. Type II receptors
– Constitutively active kinase domains (even in the absence of the bound
ligand).
– Able to phosphorylate and activate the type I receptor.

90. • Type I receptors are kept inactive by a portion of
its cytosolic domain that blocks its kinase activity.
• Type II receptors binds to, and phosphorylate,
Type I receptors. This removes the inhibition of
Type I kinase activity.
• Type I receptors then phosphorylate Smad
transcription factors which dimerize & enter
the nucleus to repress or activate target gene
expression.

91. Receptor Tyrosine Kinase
RTK ligands, such as fibroblast growth factor (FGF), epidermal
growth factor (EGF), nerve growth factor (NGF) etc. bind as dimers.
1.Ligand binding to RTK monomers results in dimer formation.
2.Receptors possess an intracellular tyrosine kinase domain. Within
the dimer the conformation is changed, locking the kinase into an
active state.
3.The kinase of one receptor then phosphorylates a tyrosine residue
contained in the second receptor.
4.Phosphorylated tyrosines function as binding sites for intracellular
signalling proteins.

93. Tyrosine Kinase Associated
Receptors
• Cytokines are the main ligands that signal
through tyrosine kinase-associated receptors.
• Like RTKs and RS/TKs these receptors
activate a cascade of phosphorylation BUT
they do not possess a tyrosine kinase domain.
• The intracellular side of each receptor is bound
to a cytosolic tyrosine kinase protein.

94. Tyrosine Kinase Associated Receptors

95. • 1. Cytokines bind simultaneously to two receptor
monomers.
• 2. This brings the two associated kinases closer
together.
• 3. One kinase phosphorylates the other kinase.
• 4. The enhanced kinase phosphorylates more tyrosine
residues on the intracellular portion of the receptor.
• 5. Phosphotyrosines serve as "docking sites" for SH2
domain-containing proteins.

98. Enzyme regulation is the process, by which cells can
turn on, turn off, or modulate the activities of various
metabolic pathways.
The four kinds of enzyme regulation are
Allosteric regulation
Reversible covalent modification
Proteolytic activation
Regulation by control proteins

99. Allosteric Regulation
Allosteric regulation is the
regulation of an enzyme or
other protein by binding an
effector molecule at the
protein's allosteric site (that is,
a site other than the protein's
active site).

103. Reversible Covalent Modification
• The activities of some regulatory enzymes are
modulated by reversible covalent
modification of the enzyme molecule.
• These include the phosphorylation,
adenylation, acetylation, uridylation, ADP-
ribosylation, and methylation of enzymes.

104. Phosphorylation
• Phosphorylation is the most common type of
regulatory modification found in eukaryotes.
• Some enzymes are phosphorylated on a single
amino acids while others are phosphorylated
at multiple sites.

107. • The attachment of phosphoryl groups is
catalyzed by protein kinases;
• Removal of phosphoryl groups is catalyzed by
protein phosphatases.
• The phosphoryl groups are attached to
serine, threonine , histidine or tyrosine
residues.

108. Adenylation
• Adenylylation : the process in which AMP is
covalently attached to a protein, nucleic acid,
or small molecule via a phosphodiester
linkage.
• In the process of deadenylylation, AMP is
removed from the adenylylated molecule.

110. Uridylation
• Uridylylation is the process in which, a
uridylyl group is introduced into a protein, a
ribonucleic acid, or a sugar phosphate,
generally through the action of a uridylyl
transferase enzyme.

112. ADP-Ribosylation
ADP-ribosylation is the addition of one or more ADP-
ribose moieties to a protein. It is observed in only a
few proteins; the ADP-ribose is derived from
nicotinamide adenine dinucleotide (NAD).

115. Proteolytic Activation
• The activation of an enzyme by peptide cleavage is
known as proteolytic activation.
• In this enzyme regulation process, the enzyme is shifted
between the inactive and active state.
• Irreversible conversions can occur on inactive
enzymes to become active.
• This inactive precursor is known as a zymogen or a
proenzyme, which is cleaved to form the active
enzyme.

118. • The cleavage is independent of ATP.
• Proteolytic activation occurs just once in an
enzyme's lifetime.
• So this type of enzyme regulation is also
termed as irreversible covalent modification.

119. Regulation by Control Molecules
• Phosphoprotein phosphatases catalyse the reverse
process of protein phosphorylation. Cyclic AMP, an
intercellular messenger, can activate protein kinase A
(PKA).
• In the absence of cAMP, Protein Kinase A (PKA)
exists as an equimolar tetramer of regulatory (R) and
catalytic (C) subunits.
• Cyclic AMP activates Protein Kinase A by altering the
quaternary structure.

121. Thank you!!