This document discusses various mechanisms of enzyme regulation in living systems. It begins by explaining that hundreds of enzyme-catalyzed reactions must be precisely controlled for proper cellular functioning. It then describes several key mechanisms by which this regulation can be achieved, including allosteric regulation, isoenzyme expression, zymogen activation, and covalent modification via phosphorylation or glycosylation. Specific examples are provided for each type of regulation, such as feedback inhibition of threonine dehydratase and phosphorylation control of glycogen phosphorylase activity. The document concludes by emphasizing that multiple regulatory strategies acting together ensure survival of the cell and maintenance of homeostasis.

1. Purnima Kartha. N
1

2. 1. Introduction
2. Allosteric Regulation.
3. Regulation through Isoenzymes.
4. Zymogen Activation
5. Covalent Modification
6. Control of Availability of enzymes
7. Conclusion
8. Reference
2

3.  In living systems hundreds of different
enzyme catalysed reactions occur
simultaneously.
 These reactions must be regulated for the
proper functioning of a living system.
 Regulatory enzymes exhibit increased or
decreased catalytic activity in response to
certain signals. 3

4. An enzyme’s catalytic activity can be
directly controlled through structural alterations
that influence the enzyme’s substrate-binding
affinity.
• Allosteric Enzyme Regulation
• Proteolytic Activation of enzymes
• Reversible Covalent Modifications
• Regulation by Isoenzymes
4

5.  Enzymatic activity can be activated or inhibited
through non-covalent interaction of the enzyme
with metabolites other than the substrate. This form
of control is termed Allosteric regulation.
 Allosteric proteins contain distinct regulatory sites
and multiple functional sites.
5

6.  Many of the ideas about ligand-
induced conformational changes
of enzymes developed as a result
of work on the biosynthetic
pathways of microorganisms.
 In 1950s, it was found that
Threonine dehydratase, the first
enzyme in the Isoleucine
biosynthesis pathway was strongly
inhibited by the end-product
Isoleucine. 6

7.  Feedback inhibition: The committed step in a
biosynthetic pathway is inhibited by the ultimate end
product of the pathway.
 The feedback inhibitor F bears little structural similarity
to A, the substrate for the regulatory enzyme E1.
 F acts on a binding site distinct from the substrate
binding site. 7

8. 8

9. Allosteric proteins show the property of cooperativity i.e.,
activity at one functional site affects the activity at others. A
slight change in substrate concentration can produce
substantial changes in activity.
Their kinetics do not obey the
Michaelis–Menten equation.
Their V versus [S] plots yield
sigmoid curves rather than
hyperbolas.
9

10.  Positive cooperativity: Ligand binding at one
site facilitates the binding of other sites on the
same molecule.
 Negative cooperativity: Ligand binding at
one site inhibits the binding of other sites on
the same molecule.
10

11.  Regulatory enzymes for which substrate and
modulators are identical are called
Homotropic.
 When the modulator is a molecule other
than the substrate, the enzyme is said to be
Heterotropic.
 Regulatory enzymes are also subject to an
activation process by a metabolite which
belongs to another metabolic pathway, which
11

12.  Allosteric enzymes typically have an
oligomeric organization.
 They are composed of more than one
polypeptide chain (subunit), and each subunit
has a binding site for substrate, as well as a
distinct binding site for allosteric effectors.
 The regulatory effects exerted on the enzyme’s
activity are achieved by conformational 12

13. CONCERTED MODEL SEQUENTIAL MODEL
13

14. ATCase :
-allosterically inhibited
CTP (feedback
inhibition)
-allosterically
activated ATP.
ATCase catalyzes the first step in the biosynthesis of
pyrimidines.
14

15.  ATCase Consists of Separable Catalytic and
Regulatory Subunits
 There are 2 catalytic trimers and 3 regulatory
dimers.
15

16.  2 distinct quaternary forms:
 T state predominates in the
absence of substrate or substrate
analogs
 R state predominates when
substrates or analogs are bound.
 The binding of the inhibitor
CTP shifts the equilibrium
toward the T state, decreasing
the net enzyme activity and 16

17.  Glycogen phosphorylase is dimer of two identical
subunits.
 ATP and Glucose-6-phosphate are negative
heterotropic effectors. ATP is a feedback inhibitor.
 AMP is a positive heterotropic effector (activator).
17

18. Isoenzymes are enzymes that differ in amino
acid sequence yet catalyze the same reaction.
18

19.  Isoenzymes are enzymes that differ in amino acid
sequence yet catalyze the same reaction.
 These enzymes display different kinetic parameters,
such as Km, or different regulatory properties.
 Encoded by different genetic loci, (arise through
gene duplication and divergence).
 Allozymes - Enzymes that arise from allelic variation
at one gene locus.
 The existence of isozymes permits the fine-tuning of
metabolism to meet the particular needs of a given 19

20.  Lactate dehydrogenase catalyses the reaction:
 In the reverse direction, it represents the last step in the
anaerobic glycolysis for the regeneration of NAD+
required for G3PDH reaction.
20

21.  The functional enzyme is a tetramer and there are 5 forms
of the enzyme.
 Human beings have two isozymic polypeptide chains for
LDH: the H isozyme highly expressed in heart and the M
isozyme found in skeletal muscle.
21

22. Zymogens are inactive precursors of enzymes.
Zymogens or proenzymes acquire full activity only upon
specific proteolytic cleavage of one or several of their peptide
bond.
Irreversible process.
22

23. • Some protein hormones
are synthesized in the
form of inactive precursor
molecules, from which the
active hormone is derived
by proteolysis.
• Insulin, an important
metabolic regulator, is
generated by proteolytic
23

24. 24

25.  The digestive enzymes are synthesised as zymogens
in the pancreatic acinar calls and stored as zymogen
granules.
 Enteropeptidase catalyses the activation of
trypsinogen as it enters the duodenum.
 Trypsin catalyses the activation of other zymogens.
 Trypsin action involves the peptide bond at the C-
terminal side of lysine or arginine side chain. 25

26.  Premature activation of zymogens is
prevented to reduce damage of the pancreas :
 by the presence of Trypsin inhibitor protein in the
pancreatic secretion
 initial trigger by enteropeptidase at a site distinct
from the site of production of zymogens.
26

27. 27

28.  The formation of blood clots - series of zymogen
activations.
 Thrombin (a serine protease) specifically cleaves
Arg–Gly peptide bonds of fibrinogen and convert it
into fibrin.
 Fibrin readily aggregates into ordered fibrous
arrays that are subsequently stabilized by covalent
crosslinks.
28

29. 29

30. The covalent attachment of a molecule can modify the
activity of enzymes and many other proteins.
A donor molecule provides a functional moiety that
modifies the properties of the enzyme.
Most modifications are reversible.
30

31. 31
Phosphorylation and dephosphorylation are the most common

32.  Some proteins such as Ras and Src are
localized to the cytoplasmic face of the plasma
membrane by the irreversible attachment of a
lipid group.
 The attachment of ubiquitin is a signal that a
protein is to be destroyed.
 Cyclins must be ubiquitinated and destroyed
before a cell can enter anaphase and proceed 32

33.  The transfer of a phosphate group from a donor to
an acceptor amino acid of a protein.
 Phosphorylation : by Kinases
 Phosphatases :remove the phosphate group
through hydrolysis of the sidechain phosphoester
bond.
33

34.  Glycogen phosphorylase, the enzyme that
catalyses the release of glucose units from
glycogen.
 Regulated by both phosphorylation and
allosteric regulation.
34

35. • Muscle glycogen phosphorylase
is a dimer of two identical
subunits.
• Each subunit contains an active
site and an allosteric effector
site near the subunit interface.
35

36. Covalent modification through
phosphorylation of Ser14 in
glycogen phosphorylase converts
the enzyme from a less active,
allosterically regulated form (the b
form) to a more active,
allosterically unresponsive form
(the a form).
36

37.  The reaction of ADP-ribosylation is catalysed by specific
enzymes, ADP-ribosyl transferases, which use NAD+ as a
substrate.
 In humans, one type of ADP-ribosyltransferases are the NAD+:
arginine ADP-ribosyltransferases, which modify amino acid
residues in proteins such as histones by adding a single ADP-
ribose group.
37

38. 38

39.  Activated by DNA cleavages –involved in DNA repair,
transformation and cellular differentiation.
 Modifies histones – causes changes in chromatin
structure.
 ADP-ribosylation modulates the activity DNA ligase
III, terminal deoxynucleotidyl transferase, α and β
DNA polymerases, topoisomerases, Ca++ and Mg++
dependant endonucleases.
 The activity of ADP-ribosyl transferase is significantly
Significance – In
Eukaryotes
39

40.  The ADP-ribosylation reactions play a role in the
toxicity of certain bacteria.
 Diphtheria toxin inhibits the EF2 elongation factor
by mono-ADP-ribosylation, which blocks protein
synthesis in the infected cell.
 Choleric and pertussis toxins provoke ADP-
ribosylation of a G protein and causes regulation of
adenylate cyclase activity, lead to increase in the
cellular ratio of cyclic AMP.
40

41.  Protein glycosylation is a post-translational
modification.
 There are two major types of glycosylation, N- and O-
glycosylations, which involve the binding of the
saccharide chain to an asparagine and a serine or a
threonine, respectively.
 N-glycosylations occur on an asparagine belonging to a
sequence Asn-X-Ser/Thr, where X can be any amino
acid except proline or aspartic acid.
 O-glycosylations occur on hydroxyl groups of a serine 41

42. 2nd step in the formation of typical O-linked oligosaccharides
in proteins such as glycophorin : A specific glycosyltransferase
catalyzes addition of a galactose residue from UDP-galactose
to C3 of N-acetylgalactosamine attached to a protein forming a
β13 linkage.
42

43.  This type of covalent modification consists of the
binding of an adenyl group to a well-defined tyrosine
residue of a protein.
 Eg: Glutamine Synthase
 Glutamine synthetase (GS) catalyses the ATP-
dependent condensation of ammonia with glutamate,
to yield glutamine.
 Bacterial GS regulated by adenylation. Mammalian
and plant GS not regulated by adenylylation.
43

44. Adenylation of glutamine
synthetase, catalyzed by the
enzyme adenylyl transferase
(ATase)
Involves the phosphodiester
bond between the OH group
of the Tyr in glutamine
synthetase and the phosphate
group of an AMP nucleotide.
44

45.  DNA ligases and RNA ligases catalyze the
formation of phosphodiester bonds at single
strand breaks with adjacent 3’ hydroxyl and 5’
phosphate termini in DNA or RNA,
respectively.
 The first step in the catalytic cycle is the
adenylylation of an active-site ε-NH3 group of
Lys residue.
45

46. 46

47. Conversion of enzyme from one form to another is
enzyme catalysed.
• Rapid change in the amount of active enzymes.
• Large amplification of the initial signal
Reversible modifications permit controlled responses
of metabolic signals.
• System is always poised for activation or inactivation.
• System can be rapidly activated, since it is reversible in nature.
• When stimulus removed, system rapidly converted back to resting
state.
47

48. As enzymes are protein in nature, they are synthesized
from amino acids under gene control and degraded
back to amino acids after its action.
Enzyme quantity depends on the rate of enzyme
synthesis and the rate of its degradation.
48

49.  Cellular physiology and differentiation
processes require that at well defined stages
the enzyme activity should start and end.
 The process of enzyme turnover necessary for
the cell to adapt to changes in the environment
and to remove an abnormal enzyme.
 The process of peptide bond hydrolysis in
enzymes do not liberate energy, unlike peptide
bond formation which utilises ATP. 49

50.  Intracellular protein degradation occur both
lysosomally and proteasomally.
 The process may be energy dependent or
independent.
 It can be selective or non selective degradation.
 Many house keeping enzymes are degraded by
lysosomal degradation.
 The short lived enzymes are degraded by extra-
lysosomal mechanisms and are present in 50

51.  Proteasomes are large multisubunit proteases that
degrade ubiquitin-tagged proteins in an ATP
dependent manner.
 Ubiquitin is a small basic peptide of 76AA found in
eukaryotic cells.
 Proteasomes degrade proteins into short peptides that
are rapidly hydrolysed by cytoplasmic exopeptidases.
51

52.  Lysosomes perform 2 processes: Phagocytosis
and Autophagy.
 Lysosomes contain different hydrolytic
enzymes which have their optimum pH at
acidic range.
 Enzymes degraded by this way do not require
ubiquitination.
52

53.  The various means by which enzyme activity are
regulated include control of catalytic activity of
enzyme and control of enzyme activity.
 Catalytic activity is regulated by mechanisms like
Allosteric regulation, Covalent Modification and
Zymogen Activation.
 Different methods of regulation ensure survival
of the cell and its proper functioning.
 Any dysfunction in the enzyme regulation
53

54.  Fundamentals of Enzymology: The Cell and
Molecular Biology of Catalytic proteins, 3rd
Edition – Nicholas. C. Price, Lewis Stevens.
 Enzymes : Biochemistry, Biotechnology, Clinical
Chemistry – Trevor Palmer.
 Enzyme catalysis and Regulation – Gordon G.
Hammes.
 Biochemistry, 5th edition – Jeremy.M.Berg, John
L. Tymoczko, Lubert Stryer.
 Biochemistry, 6th edition – Reginald.H.Garrett,
Charles. M Grisham. 54

55. Thank You!!
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