Enzyme regulation

In this slide, you can see the different types of enzyme regulation with examples.

1. Enzyme Regulation Purnima Kartha. N 1

2. Contents Introduction Allosteric Regulation. Regulation through Isoenzymes. Zymogen Activation Covalent Modification Control of Availability of enzymes Conclusion Reference 2

3. Introduction 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. Enzyme Regulation 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. Allosteric Regulation 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 A llosteric 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 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. Activation and Inhibition 8

9. Cooperativity 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. Cooperativity 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 H eterotropic . Regulatory enzymes are also subject to an activation process by a metabolite which belongs to another metabolic pathway, which leads to crossed regulation of metabolic pathways. 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 changes occurring in the protein when effector metabolites bind. 12

13. 2 models of Allostery 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 ATCase Consists of Separable Catalytic and Regulatory Subunits There are 2 catalytic trimers and 3 regulatory dimers. 15

16. ATCase 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 reducing the rate of N - carbamoylaspartate generation . 16

17. Glycogen Phosphorylase 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 for Regulation Isoenzymes are enzymes that differ in amino acid sequence yet catalyze the same reaction . 18

19. Isoenzymes for Regulation 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 tissue or developmental stage. 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. Lactate dehydrogenase ( LDH) 20

21. Lactate dehydrogenase ( LDH) 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. Zymogen Activation Zymogens are inactive precursors of e nzymes. Zymogens or proenzymes acquire full activity only upon specific proteolytic cleavage of one or several of their peptide bond. Irreversible process. 22

23. Insulin 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 excision of a specific peptide from proinsulin . 23

24. Proteolytic Enzymes of the Digestive Tract 24

25. Proteolytic Enzymes of the Digestive Tract 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. Proteolytic Enzymes of the Digestive Tract 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. Activation of Chymotrypsin 27

28. Clotting Factors 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. Clotting Factors 29

30. Covalent Modification 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. Covalent Modification 31 Phosphorylation and dephosphorylation are the most common type of modifications.

32. Covalent Modification is not readily reversible always. 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 through the cell cycle. 32

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

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

35. Glycogen Phosphorylase 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. Glycogen Phosphorylase 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. ADP- Ribosylation 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. ADP- Ribosylation 38

39. ADP- Ribosylation 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 higher in tumoral cells than in normal cells. Significance – In Eukaryotes 39

40. ADP- Ribosylation 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. Glycosylation 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 or threonine side-chain, found in a β-turn 41

42. Glycosylation 2 nd 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 β 1  3 linkage. 42

43. Adenylation This type of covalent modification consists of the binding of an adenyl g roup 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 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. Adenylation 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 -NH3 group of Lys residue. Mammalian and virally encoded ligases utilize ATP to adenylylate the lysine, whereas bacterial ligases exploit NADP +.   45

46. Adenylation 46

47. Significance of Covalent Modifications 47

48. Control of Availability of Enzyme 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. Control of Enzyme Availability 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. Enzyme Degradation 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 smaller quantities. 50

51. Proteasomal Degradation 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. Lysosomal Degradation 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 . The hydrolytic enzymes include cathepsins and endopeptidases . 52

53. Conclusion 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 pathways can lead to pathological consequences. 53

54. Reference Fundamentals of Enzymology: The Cell and Molecular Biology of Catalytic proteins, 3 rd Edition – Nicholas. C. Price, Lewis Stevens. Enzymes : Biochemistry, Biotechnology, Clinical Chemistry – Trevor Palmer. Enzyme catalysis and Regulation – Gordon G. Hammes . Biochemistry, 5 th edition – Jeremy.M.Berg , John L. Tymoczko , Lubert Stryer . Biochemistry, 6 th edition – Reginald.H.Garrett , Charles. M Grisham. Biochemistry, 4 th edition – Donald Voet , Judith.G.Voet . 54

55. Thank You!! 55