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Mechanism of Enzyme Action BCH 321 Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own,

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Presentation on theme: "Mechanism of Enzyme Action BCH 321 Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own,"— Presentation transcript:

1 Mechanism of Enzyme Action BCH 321 Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own, they can be found on: –References supplied –Atlases or –The web King Saud University College of Science Department of Biochemistry

2 2 Mechanism of Enzyme Action Proteases Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation (BCH 320) are not my own, they can be found on: –References supplied –Atlases or –The web King Saud University College of Science Department of Biochemistry

3 3 Proteases (or peptidases) Enzymes that catalyze the breaking of the amide peptide bond (proteolysis) by hydrolysis.

4 4 Proteases are essential to physiologic processes Inflammation Infection Fertilization Allergic reactions Cell growth and death Blood clotting Tumor growth Bone remodeling Etc…

5 5 Proteolytic enzymes are divided into two different categories Limited proteolysis: a protease cleaves only one or a limited number of peptide bonds of a target protein leading to the activation or maturation of the formerly inactive protein. –e.g. conversion of prohormones to hormones. Unlimited proteolysis: in which proteins are degraded into their amino acid constituents. –Ubiquitin/proteasome pathway: First conjugated to multiple molecule of the polypeptide ubiquitin. This modification marks them for rapid hydrolysis by the proteasome in the presence of ATP. –Lysosome pathway: Proteins are transferred into lysosomes which contain proteases that completely digests the protein into the amino acids. These unlimited proteolysis pathways are essential for protein quality control and the reuse of amino acids during starvation.

6 6 Some Cells Secrete proteases Proteases are secreted by cells into the surrounding tissues to cause the destruction of proteins in extracellular material. Secreted into an area (stomach) for the breakdown of protein in the diet.

7 7 Protease, peptidase, or proteinase? Protease: is synonymous with peptidase. Subclass EC 3.4.). 3 = hydrolase 4 = amide bond (peptide bond) The Peptidases are comprised of two groups of enzymes: –Endopeptidases: cleave peptide bonds at points within the protein. –Exopeptidases: remove amino acids sequentially from either N or C- terminus. –Proteinase: synonymous with endopeptidase.

8 8 The EC nomenclature for proteases 3.4.11 Aminopeptidases3.4.13 Dipeptidases 3.4.14 Dipeptidyl-peptidases and tripeptidyl peptidases 3.4.15 Peptidyl-dipeptidases 3.4.16 Serine-type carboxypeptidases 3.4.17 Metallocarboxypeptidases 3.4.18 Cysteine-type carboxypeptidases 3.4.19 Omegapeptidases 3.4.21 Serine proteinases 3.4.22 Cysteine proteinases 3.4.23 Aspartic proteinases 3.4.24 Metallo proteinases 3.4.99 Proteinases of unknown mechanism

9 9 The four mechanistic classes of proteases Serine proteases Cysteine proteases Aspartic proteases Metallo proteases

10 10 Serine protease catalytic mechanism Serine Proteases often use the chymotrypsinogen numbering: His 57, Asp 102 and Ser 195 Two distinct evolutionarily unrelated families: (The classic example of convergent evolution) Mammalian: chymotrypsin, trypsin or elastase Bacterial :subtilisin. Ser/His/Asp Catalytic triad mechanism Ser: nucleophile His: general base Asp: help orient and neutralizes charge on His Covalent tetrahedral intermediates Covalent catalysis

11 11 Famous serine proteases Trypsin This serine endopeptidase is the active form (MW 24,000) of the pancreatic proenzyme trypsinogen that is activated in the intestine by enterokinase. Chymotrypsin A group of serine proteases found in pancreatic secretions that hydrolyze peptide bonds in the process of digestion. This endopeptidase is made in the exocrine pancreas as a precursor that is activated by proteolytic cleavage in the duodenum by trypsin. Elastase MW 25.000. Serine protease of broad specificity made in the exocrine pancreas. Subtilisin Serine protease of bacterial origin. Plasmin Serine protease derived from the precursor plasminogen that breaks down insoluble fibrin thus disolving blood clots. Thrombin Serine protease present in plasma as a precursor called prothrombin (MW 72,000). The active form (MW 34,000) converts plasma fibrinogen into insoluble fibrin forming the basis of a blood clot. Kallikrein Causes the liberation of kinins from plasma protein precursors causing vasodilation and possibly hypotension. Streptokinase Protease produced by hemolytic bacteria that activates plasminogen thus producing plasmin which disolves fibrin. Therapeutically streptokinase has been used to dissolve pulmonary emboli and venous thromboses. Urokinase This protease is found in human blood and urine. It activates plasminogen by the cleavage of a propeptide forming the active serine protease plasmin. Therapeutically it is used to dissolve blood clots.

12 12 The Serine Proteases Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA All involve a serine in catalysis - thus the name Ser is part of a "catalytic triad" of Ser, His, Asp Serine proteases are homologous, but locations of the three crucial residues differ somewhat Enzymologists agree, however, to number them always as His-57, Asp-102, Ser-195 Burst kinetics yield a hint of how they work!

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14 14 Serine Protease Mechanism A mixture of covalent and general acid-base catalysis Asp-102 functions only to orient His-57 His-57 acts as a general acid and base Ser-195 forms a covalent bond with peptide to be cleaved Covalent bond formation turns a trigonal C into a tetrahedral C The tetrahedral oxyanion intermediate is stabilized by N-Hs of Gly-193 and Ser-195

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16 16 Artificial Substrate for Serine Proteases

17 17

18 18 Assay for Serine Proteases

19 19 Identification of Catalytic Residue Ser-195

20 20

21 21

22 22 (N-tosyl-L-phenylalanine chloromethylketone) Identification of Catalytic Residue His-57

23 23 1. Conformational distortion forms the tetrahedral intermediate and causes the carboxyl to move close to the oxyanion hole 2. Now it forms two hydrogen bonds with the enzyme that cannot form when the carbonyl is in its normal conformation. 3. Distortion caused by the enzyme binding allows the hydrogen bonds to be maximal.

24 24

25 25

26 26 Serine proteases Several different families - all have Ser in active site and all have the same reaction mechanism. The two most commonly-studied are: Trypsin family Subtilisin family

27 27 Trypsin family of Serine Proteases Includes: trypsin chymotrypsin (used for numbering) elastase thrombin coagulation enzymes plasmin complement C1r and C1s

28 28 Serine proteases Synthesized in zymogen (inactive) form and activated by cleavage of specific peptide bonds. Catalytic triad: Serine 195 Histidine 57 Aspartate 102 Numbering based on chymotrypsin.

29 29 Zymogen activation (Lehninger 8-31)

30 30 Chymotrypsin structure (Lehninger Fig 8-18)

31 31 Chymotrypsin Diagram from Branden & Tooze, 1991

32 32 Key residues Ser 195 (1/28) labelled with DFP (diisopropylfluorophosphate) His 57 labelled with Tosyl-Phe- chloromethyl ketone Labelling of these residues inactivates serine proteases.

33 33 Protease - key areas Binding Main chain binding (non-specific) Specificity pocket ( residue 189 ) Catalysis active site - charge relay system oxyanion hole formed

34 34 Specificity pocket explains protease specificity (residues 189, 216 and 226) Chymotrypsin Trypsin Elastase aromatic basic small, uncharged Diagram from Branden & Tooze, 1991

35 35 Substrate modification (Lehninger) Small substrates for chymotrypsin

36 36 Two domains and other key residues Active site, S195, H57 and D102, lies between domains in chymotrypsin oxyanion hole (193- 195) main chain substrate binding (214-216) substrate specificity pocket (189,216,226) Branden & Tooze, 1991

37 37 Oxyanion Hole Role Stabilizes the tetrahedral transition state Form H-bonds Uses catalytic triad plus Gly 193 Positions and orient substrate for hydrolysis Stabilizes the TS

38 38 Zymogen to active A: zymogen Chymotrypsinogen B: active chymotrypsin

39 39 Subtilisin family (SB clan) Includes furin, PC2 and PC3 - all active in prohormone processing No sequence identity with trypsin family Structure quite different (  structure; trypsin is  -barrel) But - same reaction mechanism and organisation of catalytic triad

40 40 Serine protease families - convergent evolution Trypsin family (H57, D102, S195) His Asp Ser Subtilisin family (D32, H64, S221) Asp His Ser

41 41 Chymotrypsin and subtilisin Different structures but similar active sites

42 42 Mechanism of Serine Protease

43 43 Cysteine protease catalytic mechanism Papain is the archetype and the best studied member of the family Cysteine proteases sometimes use the Papain numbering system: Cys25 and His159. Asn175 helps stabilize the Cys - and His + ion pair Unlike Ser protease Cys already ionized before substrate binding. a priori activated enzymes. Cys25 and His159 play the same catalytic roles as Ser195 and His57 respectively in the serine proteases. Covalent tetrahedral intermediates 1 2 3 4

44 44 Famous cysteine proteases Papain Thiol protease from the papaya. MW of about 23,400 used for tenderizing meats. Bromelain Thiol protease found in pinapple juice and stem tissue. Glycoprotein of about 33,000 MW used in meat tenderizing, beer production and hydrolization of proteins in the food industry. mammalian lysosomal Cathepsins (11) These intracellular cysteine proteases are involved in such diverse activities as blood clotting, cancer growth and metastasis and bone remodeling. Several varieties have been isolated from various tissues, each having specific functions. cytosolic calpains (calcium-activated) parasitic proteases (e.g Trypanosoma). Interleukin-1-beta Converting Enzyme

45 45 Aspartic protease catalytic mechanism Bi-lobe enzymes with the active site located between two homologous lobes. Asp general acid-base catalysis 2 "push-pull" mechanisms ( steps 1 and 3) Non-covalently bound (neutral) tetrahedral intermediate. Low-barrier H-bonds may be involved. 1 3

46 46 Famous aspartic proteases Pepsin Aspartic protease that cleaves bonds involving phenylalanine and leucine preferentially. MW 34,500. This endopeptidase is the principal digestive enzyme in gastric juice formed from the precursor pepsinogen (MW 38,000). (first protein crystals to diffract) Renin Also known as angiotensinogenase. It is made by the juxtaglomerular cells in the kidney, released into the blood stream and acts to convert angiotensionogen into angiotensin I. It is an aspartyl protease having a molecular weight of about 40.000 Renin is elevated in some forms of hypertension.. cathepsins D fungal proteases (penicillopepsin, rhizopuspepsin, endothiapepsin). viral proteinases such as HIV protease (retropepsin).

47 47 The Aspartic Proteases Pepsin, chymosin, cathepsin D, renin and HIV-1 protease All involve two Asp residues at the active site Two Asps work together as general acid-base catalysts Most aspartic proteases have a tertiary structure consisting of two lobes (N-terminal and C-terminal) with approximate two-fold symmetry HIV-1 protease is a homodimer

48 48

49 49 Aspartic Protease Mechanism The pK a values of the Asp residues are crucial One Asp has a relatively low pK a, other has a relatively high pK a Deprotonated Asp acts as general base, accepting a proton from HOH, forming OH - in the transition state Other Asp (general acid) donates a proton, facilitating formation of tetrahedral intermediate

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51 51 Asp Protease Mechanism What evidence exists to support the hypothesis of different pK a values for the two Asp residues? Bell-shaped curve is a summation of the curves for the two Asp titrations In pepsin, one Asp has pK a of 1.4, the other 4.3

52 52

53 53

54 54 Mechanism of Aspartic protease

55 55 HIV-1 Protease A novel aspartic protease HIV-1 protease cleaves the polyprotein products of the HIV genome This is a remarkable imitation of mammalian aspartic proteases HIV-1 protease is a homodimer - more genetically economical for the virus Active site is two-fold symmetric Two Asp residues - one high pK a, one low pK a

56 56

57 57 Therapy for HIV? Protease inhibitors as AIDS drugs If the HIV-1 protease can be selectively inhibited, then new HIV particles cannot form Several novel protease inhibitors are currently marketed as AIDS drugs Many such inhibitors work in a culture dish However, a successful drug must be able to kill the virus in a human subject without blocking other essential proteases in the body

58 58

59 59

60 60 Metallo-protease catalytic mechanism Most contain a Zn atom that is involved in catalysis. Sometimes Zn can be replaced by Co or Ni. Zn is usually coordinated by 2 His and 1 Glu. Many contain the sequence HEXXH Non-covalent (oxyanion) tetrahedral intermediate. After the attack of a Zn-bound water molecule on the carbonyl group of the scissile bond. The intermediate breaks down by transfer of a proton From a Glu to the leaving group (not shown). H

61 61 Famous metallo-proteases Thermolysin Extracellular thermostable zinc metalloprotease from bacterial sources. Collagenase Proteases that breakdown collagen that are produced by some species of bacteria such as Clostridium. Also isolated in human wound tissue, skin, bone, leukocytes, and cornea. Family of metalloproteases (MW 68,000- 125,000) that require zinc and calcium for activity. They are used in research for the isolation of cells from animal tissues.

62 62 Mechanism of Thermolysin

63 63 Zn 2+ binds the amide carbonyl group Zn 2+ polarizes the carbon of carbonyl group The nucleophile water (OH - ) is activated by Glu-143 His-231 is the acid for protonating the leaving group (R 1 -NH 2 ) Mechanism of Thermolysin

64 64 The Schechter and Berger nomenclature for the description of protease subsites

65 65 Scissile bond and Subsite nomenclature S1 S3 S1 peptidase P1 P2 P3 P1’ scissile bond I86 I144 S88 D142 V132 I101 I86 F84 P87 S90 M91 L95 I144 Y143 K145 solvent

66 66 Nucleophilic attack from Si vs. Re face re-face Ser/His/Asp proteases si-face Ser/Lys protease 8 76

67 67 Angle of nucleophilic attack on a carbonyl The Dunitz-Bürgi angle (105°) From analysis of small molecule high resolution crystal structures it was observed that: The preferred O … C=O angle is 105° 105  From inhibitor and substrate co-crystal structures: The nucleophiles in the active-site of proteases (Ser hydroxyl O , Cys Thiol S , waters) Are often orientated with respect to the scissile carbonyl close to the Dunitz-Bürgi angle (105°) Ser

68 68 Evolutionary Classification of proteases MEROPS: Protease Data BaseMEROPS –Families: Proteases with statistically significant similarities in amino acid sequence. –Clans: Protease families that are thought to have common evolutionary origins.

69 69 Proteases as research tools Limited proteolysis / sequencing / MS –Protein crystallization –Protein mobility (dynamics) –Protein-Protein interactions NickPred: Web tool to understand limited proteolysis using PDBsNickPred: PROWL: A collection of protocols for limited proteolysis combined with Mass SpecPROWL:

70 70 Mechanism of Electron Transfer in Biological System 1.Two One Electron Steps Mechanism 2.One Two Electron Steps Mechanism

71 71 Two One Electron Steps Mechanism A free-radical intermediate must form which highly reactive and very energetic species Discrete long-lived radical intermediates are infrequently seen in biological systems Examples: flavin-Coenzyme Vit C, E, K, CoQ Metalloenzymes

72 72 1.Hydride (H - :) Mechanism 2.H+ Abstraction Mechanism One Two Electron Steps Mechanism Most of enzyme hydrogenases are enzymatic dehydrogenation where H - ion transfer

73 73 One Two Electron Steps Mechanism Hydride Mechanism Carbonium Ion Carbonanion Ion Proton Abstraction Mechanism

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