11 proteases.ppt

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  • The peptides A and B are removed by proteolytic digestion.
  • Amide bonds between different fibrim molecules.
  • 11 proteases.ppt

    1. 1. THE PROTEASES3 big problems for the kinetics:H2O is a bad nucleophileNH is a bad leaving grouploss of resonance during reactionNo problem for thermodynamics ( ∆G << 0 )NNHO HHOHH2O:NOOHOHH3NH- +∆G < 0
    2. 2. NNHO HHOHH2OHydrolyzed under very harsh conditions,in acid (HCl 6 M, 110°C, 24-72 h)or base (KOH 1 M 100°C 24 h)The peptide bond is « stable » under physiological conditionst1/2 102years despite its thermodynamic instabilityTHE PROTEASES
    3. 3. 5 biological strategies to solve the problems, 5 classes of proteases1. Serine proteasesTrypsin in mammalian digestionCoagulation factors (Thrombine)2. Cysteine proteasesPapaineCathepsines (in the lysosomes)3. Aspartyl proteasesPepsine (in our stomach)AIDS virus protease4. Metal ion proteases (Zn2+)Carboxypeptidases5. Threonine proteasesProteasomeNNHO HHOHH2OTHE PROTEASES3 big problems for the kinetics:H2O is a bad nucleophileNH is a bad leaving grouploss of mesomery during reaction
    4. 4. Where the proteases act?Exo-proteases (exo-peptidases, cut amino acids fromthe N- or from the C-terminal of proteins/peptides)Endo-proteases (cut in the interior of proteins/peptides)SpecificityNon-specific (Proteinase K, used for stability studies of proteins)Specific proteases (Trypsine X-X-Arg↓X-X et X-X-Lys↓X-Xrecognize 1 side chainVery specific (Thrombine LeuValProArg↓GlySer )recognize 6 side chainsTHE PROTEASES
    5. 5. IMPORTANT HINT! The protease best substrates are UNFOLDED proteins.Compact protein domain are not hydrolyzed, instead the connecting domainsare hydrolyzed  Essential application in protein biochemistry and imunologyfor domain preparation by controlled proteolysisClassical experiment: Porter 1955, preparation of immunoglobulin fragmentsby treatment with papain or pepsinTHE PROTEASESDisulfide bonds
    6. 6. THE PROTEASESSDS gel electrophoresis
    7. 7. Stanley B. Prusiner• strange pathogen (resistance to UV, heat etc…)•transmission to mouse (incubation time 150-300 days)• 1975-77: transmission to hamster (70 days)P r PCP r P s e n P r P 2 7 - 3 0P r P r e sP r PS C1 2 3 2infectedinfectedproteinproteinasease KKThe presence of the Prion protein is demonstrated by theresistence to proteolysis
    8. 8. Blood clottingBlood clotting results from a cascade of reactions.In a cascade, a signal initiates a series of steps, each of themcatalyzed by an enzyme. At each step the signal is amplified.In blood clotting the activated form of one clotting factorcatalyzes activation of the next.Very small amounts of the initial factors trigger the cascade,=> rapid response to trauma (e.g., damage to a blood vessel).
    9. 9. Blood clottingFibrin fiber
    10. 10. Conversion of fibrinogen to fibrin causes clotting.The final step of clotting is conversion of fibrinogen to fibrin bythrombin, a protease.Fibrinogen has 6 protein chains (2x Aα, Bβ and γ), folded intoglobular units connected by rods. Thrombin cleaves 4peptides from the Aα and Bβ chains in the central globule,resulting in fibrin monomer (αβγ)2.
    11. 11. Carboxyl ends of the β- and γ chains interact with the newlyexposed N-terminal regions => polymerization (protofibrils).
    12. 12. Blood CoagulationHemostasis
    13. 13. Fibrils are stabilized by cross-linking: formation of amidebonds between lysine and glutamine by transglutaminase,which is activated from protransglutaminase by thrombin.The network of fibrils forms the clot.
    14. 14. Activation of thrombin.Thrombin activates fibrinogen, but how is thrombin activated ?Thrombin is activated by proteolytic activation of prothrombinwith factors Xa (also a protease) and Va. Activation removes agla and 2 kringle domains.Modular structure of prothrombin
    15. 15. Use of chromogenic substrates for studying the proteasesThrombine (enzyme in blood coagulation)Natural substrate: le fibrinogen (a large protein, about 2000 residues)Benzoyl-Phe-Val-Arg ↓NH NO2NH2 NO2The product (p-nitro-aniline) est yellow(λ 380 nm)
    16. 16. 1. The serine proteasesProteases having an essential serine in the active siteProtéasesTrypsineChymotrypsineElastaseSubtilisine (Bacilus subtilis)Same mechanism for esterasesLipasesEsterases(acétyl)choliesteraseNOO HHOHH2OAmides and esters have similarstructure and reactivityNNHO HHOHRH2OON
    17. 17. Identification of active serine in serine proteasesAn Unusually Reactive Serine in ChymotrypsinChymotrypsin is inactivated by treatment withdiisopropylphosphofluoridate (DIPF), which reacts only with serine195 among 28 possible serine residues. No reaction with theunfolded enzyme, nor with free serine
    18. 18. Identification of active serine in serine proteasesReaction timePercentInhibitionofactivity(%)100500No substrateAdd substrateS+ DIFP+ DIFP & substrateXXAddition of Substrate protects DIFP Inhibition
    19. 19. substrate inactivator(TPCK)With [14C]TPCK get 1 equiv. [14C] bound; pepsin hydrolysisgives a [14C] peptide with His-57 modifiedCH2 CHNHSO2CCH3CH2 CHNHSO2CCH3OCH3O OCH2Cl2.11 2.12Evidence for Histidine Participation
    20. 20. The serine proteases: the specificity pocket
    21. 21. 4 Catalytic elements in serine proteasesO-
    22. 22. Specificity pocketAa 189, 216, 226Oxyanion holeAa 193-195Substrate bindingAa 214-216Catalytic triadSer195, His57, Asp1024 Catalytic elements in serine proteasesChymotrypsin
    23. 23. ChymotrypsinSTRUCTURE: David BLOW 1968
    24. 24. Serine is the NUCLEOPHILEHistidine is a BASE: it binds the serine’sproton and decreases its pKa from 15 toabout 7The aspartate keeps the histidine in thecorrect orientation (an old theory: protonrelay, but the proton does not move):
    25. 25. An example of CONVERGENT evolutionTrypsinSubtilisinbut identical active site!
    26. 26. Évolution convergenteCONVERGENT evolution
    27. 27. Thrombin and Chymotrypsin areHOMOLOGS(Almost identical structures, similar sequences
    28. 28. Evolution is most often DIVERGENTA few examples ofCONVERGENT evolution« Ancestral » gene, duplicationand separate evolution by mutationTrypsine, chymotrypsine, élastaseStructure très similaireFamille de protéinesTriade: Ser195, His57, Asp102SubtilisineStructure très différenteTriade: Ser221, His64, Asp32Different genes, protein evolutionTo a similar active site configuration
    29. 29. Many serine proteases age activated by proteolysis(protection of the cells which synthetize the proteases)
    30. 30. ActivesiteresiduesHydrophobicpocketSerine Protease Mechanism - Chymotrypsin
    31. 31. Disulfide bridges
    32. 32. This is a reactionINTERMEDIATEand not a transitionstateReaction coordinate
    33. 33. The C-terminal part of thesubstrate dissociated andleaves the Acyl-enzyme
    34. 34. STEP 2: Acyl-enzymehydrolysis
    35. 35. Kinetic demonstration of the serine protease mechanism: burst kinetics
    36. 36. Demonstration of the serine protease mechanism: site-directed mutagenesisNature. 1988, 332(6164):564-8.Substrate: N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilideBacillus amyloliquefaciens subtilisin, these functional elementsimpart a total rate enhancement of at least 109to 1010times thenon-enzymatic hydrolysis of amide bonds
    37. 37. Reaction mechanism of a serine protease(in this case, subtilisin)Note the three residues of the “catalytic triad”: Ser221, His64, & Asp32.
    38. 38.
    39. 39. Subtilisine Km kcat/Km kcatAsp32 H64 S221 (µM) (M-1s-1) s-1Asp His Ser 220 250000 55Ala His Ser 480 5 0.0024Asp Ala Ser 390 0.1 0.000039Asp His Ala 420 0.1 0.000042Ala Ala Ala 420 0.1 0.000042kSerHisAsp/knon-enzymatique = 3 750 000 000kAlaAlaAla/knon-enzymatique = 3 000Demonstration of the serine protease mechanism: site-directed mutagenesis
    40. 40. 1. When very low residual activities are expected, a very low levelof contamination with other proteases is a serieus problem.How has this been avoided? Serine24 (on the protein surface)has been replaced by a Cysteine which makes possible proteinpurification by covalent affinity chromatography.2. A second problem could be the mis-incorporation duringtraduction. An error rate of 1/1000 can be a problem !
    41. 41. 3. Ascertaining the role of specific amino acids in catalysis bysite-directed mutagenesis can easily by interpreted if thechemical step is rate-limiting (A). If the substrate binding is rate-limiting (B), it is well possible to miss important details of themechanism.E + SESE + PGReaction coordinate∆G∆GE + SESE + PGReaction coordinate∆G∆GA BReplacing an active-site residue will slown down reaction in A butnot in BThe measured rate is slower with the mutantNo apparent effect!
    42. 42. Take home lesson: even with no catalytic residues, the enzyme stillaccelerates the reaction better than 1000-fold the rate of theuncatalyzed reaction. Way to bind that transition state!
    43. 43. Demonstration of the serine protease mechanism: site-directed mutagenesisSite-directed mutagenesis and the role of the oxyanion hole in subtilisin.Bryan P, Pantoliano MW, Quill SG, Hsiao HY, Poulos T.Proc Natl Acad Sci U S A. 1986 Jun;83(11):3743-5.Reaction intermediate isstabilized by main-chain NHin chymotrypsin: its rolecannot be probed by site-directed mutagenesisReaction intermediate isstabilized Asn side-chain insubtilisin: its role CAN beprobed by site-directedmutagenesis!!!
    44. 44. Demonstration of the serine protease mechanism: site-directed mutagenesisIn the transition state complex, the carbonyl group of the peptide bond to be hydrolyzedis believed to adopt a tetrahedral configuration rather than the ground-state planarconfiguration. Crystallographic studies suggest that stabilization of this activatedcomplex is accomplished in part through the donation of a hydrogen bond from theamide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. Tospecifically test this hypothesis, leucine was introduced at position 155. Leucine isisosteric with asparagine but is incapable of donating a hydrogen bond to thetetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but agreatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayedwith a peptide substrate. These kinetic results are consistent with the Asn-155mediating stabilization of the activated complex and lend further experimental supportfor the transition-state stabilization hypothesis of enzyme catalysis.
    45. 45. A recent addition to the serine protease mechanism:the Low barrier hydrogen bonds2.8 Å 2.55 Å 2.29 Å
    46. 46. A recent addition to the serine protease mechanism: the Lowbarrier hydrogen bondsFormation of a short (less than 2.5 angstroms), very strong, low-barrier hydrogen bond in the transition state, or in an enzyme-intermediate complex, can be an important contribution to enzymiccatalysis. Formation of such a bond can supply 10 to 20 kilocaloriesper mole and thus facilitate difficult reactions such as enolization ofcarboxylate groups. Because low-barrier hydrogen bonds form onlywhen the pKas (negative logarithm of the acid constant) of theoxygens or nitrogens sharing the hydrogen are similar, a weakhydrogen bond in the enzyme-substrate complex in which thepKa’s do not match can become a strong, low-barrier one if thepKa’s become matched in the transition state or enzyme-intermediate complex.Low-Barrier Hydrogen Bonds and Enzymic CatalysisW. W. Cleland and Maurice M. Kreevoy
    47. 47. A second recent addition to the serine protease mechanism:Substrate assisted catalysis
    48. 48. A recent addition to the serine protease mechanismSubstrate assisted catalysis
    49. 49. A second recent addition to the serine protease mechanismSubstrate assisted catalysis
    50. 50. A recent addition to the serine protease mechanismSubstrate assisted catalysis
    51. 51. Can proteases be used for protein SYNTHESIS?CHEMICAL ligation
    52. 52. Kaiser and co-workers demonstrated the practicality of this workby preparing a subtilisin variant, thiolsubtilisin, where the activesite Ser was chemically converted to Cys (S221C)) Usingactivated esters to acylate the active site Cys in the presence ofamine nucleophiles, it was possible to efficiently synthesizeamide bonds. The ratio of aminolysis to hydrolysis is 600-foldgreater for thiolsubtilisin relative to subtilisin; the variantselenolsubtilisin was later prepared by Hilvert and co-workers andshown to be 14,000-fold more effective for aminolysis thansubtilisin.
    53. 53. Aminolysis/HydrolysisSerine OH 1.0Cysteine SH 600Selenocysteine SeH 14000 Meth Enz 289, 298-313Subtiligase: a tool for semisynthesis of proteins.Chang TK, Jackson DY, Burnier JP, Wells JA.Department of Protein Engineering, Genentech, Inc., South San Franc94080.AminolysisHydrolysis
    54. 54. Serine hydrolases: proteases and other enzymesAsparaginaseSerine proteasesEsterasePenicillin acylaseβ-lactamase
    55. 55. The acetyl-cholinesterase – a serine esterase
    56. 56. Acetylcholinesterase: an archetype for cation–p bonding in biology?Acetylcholinesterase is often considered as the foremost example ofcation–p bonding in biological molecular recognition. In its interactionwith acetylcholine, it serves as an excellent model for the recognition ofquaternary amines by proteins. Early kinetic, spectroscopic andchemical modification studies [17] suggested that the active site ofacetylcholinesterase is divided into two subsites: the esteratic site (thesite of bond breaking/making) and the anionic (choline binding) site.The anionic site is a misnomer, as this site is in fact uncharged andlipophilic. The molecular detail of acetylcholinesterase was revealedfollowing the determination of the crystal structure of the enzyme fromTorpedo californicans [18]. A structure for the enzyme–substratecomplex is not available, but the details of substrate binding can beextrapolated from the structure of the enzyme alone [18] and those ofthe enzyme complexed with tacrine, edrophonium and decamethonium[19].
    57. 57. 2. Cysteine proteasesPapaïne from plants is one exampleCathepsines (protease from lysosomes)SH N N H:
    58. 58. Protease from AIDS virus: an aspartyl protaase3. Aspartyl proteases
    59. 59. 3. Aspartyl protéasesPepstatin is a potent inhibitor of aspartyl proteases. It is a hexa-peptide containing the unusual amino acid statine (Sta, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid), having the sequenceIsovaleryl-Val-Val-Sta-Ala-Sta (Iva-Val-Val-Sta-Ala-Sta). It wasoriginally isolated from cultures of various species of Actinomycesdue to its ability to inhibit pepsin at picomolar concentrations. Itwas later found to inhibit nearly all acid proteases with highpotency and, as such, has become a valuable research tool, aswell as a common constituent of protease inhibitor cocktails.This is a TRANSITION STATE ANALOGIsovaleryl-Val- Val- Sta- Ala- Stastatine
    60. 60. carboxypeptidase A4. Metallo-proteases
    61. 61. During proteasome-catalysed transpeptidation, the energy from peptide-bond hydrolysis fuels subsequentpeptide-bond ligation. When presented with the three- and six-residue components of the nine-residue peptide,the proteasome was unable to splice them together. However, when supplied with the six and seven-residuefragments that comprise the 13-residue precursor peptide, the proteasome efficiently produced the nona-peptide.These observations indicated that the proteasome can catalyse peptide-bondformation only when the process is linked to peptide-bondhydrolysis. Nucleophilic attack of peptide bonds by the hydroxylgroup of an active-site threonine in the proteasome results in anacyl-enzyme intermediate, in which the peptide and the threonineare joined by an ester bond. The acyl-enzyme intermediate playsa part in the proteasome-catalysed transpeptidation event. In thefirst step the hydroxyl group of an active-site threonine catalysesthe cleavage of a precursor peptide, generating an N-terminal anda C-terminal fragment. In the second step an active-site threonineattacks the peptide bond in the N-terminal fragment forming anacyl-enzyme intermediate with the N-terminal peptide. At this pointthe N-terminus of the C-terminal peptide fragment attacks theacyl-enzyme intermediate and, recycling the energy from thecleavage reaction, ligates onto the (now cleaved) N-terminalpeptide. This transpeptidation model explains how peptide-bond hydrolysis and formation occur togetherwithout the net input of energy. It shows also that the splice site need not be highly conserved because, once apeptide bond has been activated at the protease active site, ligation of almost any incoming peptide with a free N-The architecture of the central chamber of the proteasome defines the catalytic specificity and also mightregulate the incidence of splicing. The substrate-binding sites that flank the scissile bond favour certain aminoacids and, therefore, enable certain peptides to linger in the active-site cavity, thus providing an opportunity foran N-terminal nucleophile to attack the acyl-enzyme intermediate. The determinants for protease-catalysedsplicing are certainly finely controlled because the active site also must enable normal proteolytic events to occur.The question that arises in the case of proteasome-catalysed protein splicing is whether the splicing process isfavoured for a functional purpose of the resulting peptides. Proteasomes may not only mediate the completedegradation of proteins, but also the processing of precursors into mature, active proteins.5. A «new» mechanism: the threonine protease in the proteasome
    62. 62. Protein Splicing: Analogy to RNA SplicingAttention: this is different from typical « enzyme » in that it issingle turn-over!
    63. 63. 1. Protein splicing is catalyzed entirely byamino acid residues contained in theintein.2. Protein splicing is an intramolecularprocess (usually).3. Protein splicing requires no coenzymes orsources of metabolic energy and thereforeinvolves bond rearrangements rather thanbond cleavage followed by resynthesis.Annu Rev Biochem. 2000;69:447-96. Protein splicing and related forms of protein autoprocessing. Paulus H.Properties of protein splicing
    64. 64. What do they look like?Small inteins are about 150 amino acids.(the smallest is 134 amino acids, largest is 1650)
    65. 65. Step 1: formation of a linear ester intermediate by NO orNS acyl rearrangement involving the nucleophilic aminoacid residue at the N-terminal splice junction;Step 2: formation of a branched ester intermediate bythe attack of the nucleophilic residue at the C-terminalsplice junction on the linear ester intermediate;Step 3: cyclization of the asparagine residue adjacent tothe C-terminal splice junction, coupled to cleavage ofthe branched ester intermediate to yield an excisedintein with a C-terminal aminosuccinimide residue andthe two exteins joined by an ester bond;Step 4: spontaneous hydrolysis of the aminosuccinimideresidue and rearrangement of the ester linking theexteins to the more stable amide bond.Annu Rev Biochem. 2000;69:447-96.Protein splicing and related forms of protein autoprocessing.Paulus H.The last step is spontaneous and irreversible.The first three steps are catalyzed by the intein
    66. 66. Dawson PE, Muir TW, Clark-Lewis I, Kent SB.Synthesis of proteins by native chemical ligation.Science. 1994 Nov 4;266(5186):776-9.Same chemistry as protein splicing has been used forspontaneous (non-enzymatic) peptide ligation
    67. 67. Proposed mechanism of amide, true peptide, and ester bond hydrolysis by proteasomes andmechanism of their inactivation by irreversible inhibitors.Kisselev A F et al. J. Biol. Chem. 2000;275:14831-14837
    68. 68. INHIBITORS
    69. 69. Une des composantes de la tri-thérapie est un inhibiteur de laProtéase du virus du SIDA, un analogue de l’état de transition3. Aspartyl protéases
    70. 70. Access to the active site of acetylcholinesterase is via a deep and narrow gorgup about 40% of the surface of the gorge) and other residues. The gorge is 20the surface of the gorge are highly conserved in acetylcholinesterases from disubstrate acetylcholine at the base of the gorge reveals the esteratic and cholesteratic site, a catalytic triad and putative oxyanion hole have been identifiedacetylcholine suggests that it forms a cation–p bond with Trp-84 in the anionicremarkable feature of acetylcholinesterase is the preponderance of aromatic rchemical character of the gorge leads to the question of its function in contribuand catalysis. Sussman and colleagues suggested two mechanisms by whichincreased [18]. First, the high hydrophobicity of the gorge produces a low dieleto enhance the effective local charge contributed by the small number of acidielectrostatically steer substrate to the active site. In the second scenario, theaffinity sites for the substrate (in particular, the choline moiety), and guides theBecause of the reduction-in-dimensionality, the rate of substrate binding is incinteractions may, therefore, have a major role to play in directing the substratesubstrate complex, whereas stronger cation–p bonding is presumably responsacetylcholine in the enzyme–substrate complex. Given the wealth of cation–pthe enzyme no doubt will remain a principal target for investigating these interInterestingly, chemical modification studies of the nicotinic acetylcholine recepresidues are located in the acetylcholine-binding site in this molecule [20,21].
    71. 71. Leupeptin, also known as N-acetyl-L-leucyl-L-leucyl-L-argininal, is a protease inhibitor that also acts as an inhibitorof calpain.It is often used during in vitro experiments when a specificenzymatic reaction is being studied. When cells are lysedfor these studies, proteases, many of which are containedwithin lysosomes, are released. These proteases, if freelypresent in the lysate, would destroy any products from thereaction being studied, and make the experimentuninterpretable. For example, leupeptin could be used in acalpain extraction to keep calpain from being hydrolyzed byspecific proteases. The suggested concentration is 1-10 µM(0.5-1 µg/ml).Leupeptin is an organic compound produced by
    72. 72. Une des composantes de la tri-thérapie est un inhibiteur de laProtéase du virus du SIDA, un analogue de l’état de transition3. Aspartyl protéases

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