Presentation is loading. Please wait.

Presentation is loading. Please wait.

Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

Similar presentations


Presentation on theme: "Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease."— Presentation transcript:

1 Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease family Serine protease activity regulation

2 Transition state theory of chemical catalysis The rate of the reaction A B is limited by the rate of formation of the transition state A with G = H - T S Energy (G) Reaction coordinate B A A G G H : transition state stabilization A S : reduction of entropy loss by non-covalent substrate binding acid, alkaline, electrophilic, nucleophilic catalysis... The entropic advantage of unimolecular over a bimolecular reaction In solution catalysis :

3 Intramolecular catalysis and effective concentration Intra-molecular reaction k 1 = 0.02 s -1 V transfert = k 1 [ acyl ] Inter-molecular reaction ++ k 2 = M -1 s -1 V transfert = k 2 [ acyl ] [ carboxyl ] Carboxyl effective concentration : k 1 / k 2 = M

4 Chemical catalysis by proteins (enzymes) Enzyme catalysis mechanisms Non-covalent substrate binding ( binding site) Transition state stabilization ( catalytic site) Reaction pathways of lower energy ( co-enzyme) enormous catalytic efficiency substrate specificity chemical energy transfer (energy coupling) regulated catalytic activity General features

5 substrate(s) binding site(s) catalytic site Product(s) release required to initiate a new catalytic cycle Flexible set of conformational states Structural features example : hexokinase P glucose + ATP P glucose-6-P + ADP P

6 Hypothesis Michaelis-Menten model of enzyme kinetics Scheme k on k off k cat E + SE.SE + P where Michaelis-Menten equation Chemical processes Conservation equations Steady state [E] 0 << [S] 0

7 How enzymes are studied ? colored or fluorescent substrates or products : spectrophotometric methods radioactive substrates and products : filtration methods reaction coupling Kinetic methods time Product concentration Initial rate saturation initial rate v variable substrate concentration very low enzyme concentration specificity controls Activity measurements without enzyme with enzyme

8 Graphical representations of Michaelis-Menten equation where Michaelis-Menten equation Maximum rate V max Michaelis constant KMKM V max KMKM V max /2 Direct representation V [S] Eadie-Hofstee V V/[S] V max -K M 1/V Lineveawer-Burk 1/V max -1/K M 1/[S]

9 The significance of Michaelis-Menten parameters Catalytic constant or turnover k cat : number of substrate molecules processed per enzyme molecule and per second Michaelis constant K M : substrate concentration at which half of the enzymes bind a substrate molecule (and V = V max /2) The specificity constant k cat /K M determines the specificity for competing substrates k cat /K M < k on < k diffusion M -1.s -1 V = [E] [S] k cat /K M For two competing substrates

10 Competitive and non-competitive inhibition In the case of reversible inhibition Non-competitive inhibition : regulation site of the enzyme catalytic activity V max decreases in the presence of inhibitor K M inchangé Competitive inhibition : the inhibitor is alike the transition state V max unchanged K M increases in the presence of inhibitor

11 Effect of inhibitors on enzyme kinetics V max KMKM V max /2 Direct representation V [S] Eadie-Hofstee plot V V/[S] V max -K M 1/V Lineveawer-Burk plot 1/V max -1/K M 1/[S] Competitive inhibition V max unchanged Non-competitive inhibition K M unchanged

12 Serine protease family Artificial substrate COOH R1 R2 HO R2OCR1 O ester acidalcohol Native substrate CCOOH H R1 H2NH2NCCOOH H R2 H2NH2NCCOOH H R2 NCC H R1 H2NH2N O H peptideCarboxyl partAmino part Serine protease specificity ProteaseR1 Chymotrypsinlarge hydrophobic amino-acids: Tyr, Trp, Phe, Met Trypsinlarge positively charged amino-acids: Lys ou Arg, but His Elastasesmall hydrophobic amino-acids : Ala

13 1 50 ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN tryp_bovin V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQ el1_pig...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQN thrb_human GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDR klkb_rat.SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPS fa9_human.NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEK fa10_bovin...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN tryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLN el1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAG thrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELD klkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMS fa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYN fa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN tryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTS el1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQ thrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKG klkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV..GSTCLASG WSSTKPLEWE fa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGR fa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV tryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVV el1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLH thrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFV klkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLL fa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHV fa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN tryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASN el1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASN thrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI... klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKEN fa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE..... fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *

14 Serine protease : practical study Stopped-flow experiment enzyme substrate Measur- ment chamber Time (msec) p-Nitrophenol « burst » p-Nitrophenyl acetate CCH 3 O O NO 2 p-Nitrophenol OH NO 2 CCH 3 O HO acetate

15 Detection of a covalent intermediate... Time (msec) p-Nitrophenol « burst » CCH 3 O O NO 2 OH NO 2 E + S Chymotrypsin + + CCH 3 O HO CCH 3 O O Chymotrypsin E.SE-P 2 E P1P1 P2P2 Energy (G) Reaction coordinate E + P 1 + P 2 E + S E-P 2 + P 1

16 … bound to serine 195 Irreversible inhibition of serine proteases by DIPF + CH 2 OH Chymotrypsin 195 CCH 3 H O H3CH3C P OF C H O H3CH3C + HF CCH 3 H O H3CH3C P O C H O H3CH3C CH 2 O Chymotrypsin 195 di-isopropyl- phosphofluoridate

17 1 50 ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN tryp_bovin V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQ el1_pig...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQN thrb_human GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDR klkb_rat.SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPS fa9_human.NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEK fa10_bovin...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN tryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLN el1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAG thrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELD klkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMS fa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYN fa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN tryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTS el1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQ thrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKG klkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV..GSTCLASG WSSTKPLEWE fa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGR fa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV tryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVV el1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLH thrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFV klkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLL fa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHV fa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN tryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASN el1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASN thrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI... klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKEN fa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE..... fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *

18 Histidine 57 is also part of serine protease catalytic site Irreversible inhibition of chymotrypsin byTPCK + Chymotrypsin 57 tosyl-L- phenylalanine chloromethyl ketone NH CH 2 C CH HC + HN CC H CH 2 NH SOO O CH 2 Cl Chymotrypsin 57 CH 2 ClCH 2 N C CH HC N Specificity group Reactive group

19 1 50 ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN tryp_bovin V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQ el1_pig...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQN thrb_human GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDR klkb_rat.SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPS fa9_human.NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEK fa10_bovin...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN tryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLN el1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAG thrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELD klkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMS fa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYN fa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN tryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTS el1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQ thrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKG klkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV..GSTCLASG WSSTKPLEWE fa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGR fa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV tryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVV el1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLH thrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFV klkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLL fa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHV fa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN tryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASN el1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASN thrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI... klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKEN fa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE..... fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *

20 The hydrogen bond network at the serine protease catalytic site: Serine 195, Histidine 57 and Aspartate 102 catalytic triad or charge relay system His 57 N C CH HC HN NH C CH HC + HN His 57 CH 2 HOSer 195 CO-O- O Asp 102 CH 2 -O-OSer 195 CO-O- O Asp 102 NH C CH HC N His 57 CH 2 -O-OSer 195 COH O Asp 102

21 1 50 ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN tryp_bovin V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQ el1_pig...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQN thrb_human GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDR klkb_rat.SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPS fa9_human.NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEK fa10_bovin...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN tryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLN el1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAG thrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELD klkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMS fa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYN fa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN tryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTS el1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQ thrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKG klkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV..GSTCLASG WSSTKPLEWE fa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGR fa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV tryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVV el1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLH thrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFV klkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLL fa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHV fa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN tryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASN el1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASN thrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI... klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKEN fa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE..... fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *

22 Serine protease mechanisms Substrate His 57 N C CH HC HN CH 2 HO Ser 195 CO-O- O Asp 102 R2NCR1 O H E + S NH C CH HC + HN His 57 CH 2 O Ser 195 CO-O- O Asp 102 R2NCR1 O-O- H Transition state E.S Covalent intermediate CH 2 O Ser 195 R2N CR1 H O His 57 N C CH HC HN CO-O- O Asp 102 H E-P 2 + P 1 Leaving group

23 Serine protease mechanisms Water molecule activation CH 2 O Ser 195 O CR1 H O His 57 N C CH HC HN CO-O- O Asp 102 H E-P 2 Transition state NH C CH HC + HN His 57 CH 2 O Ser 195 CO-O- O Asp 102 OCR1 O-O- H E.P 2 CH 2 HO Ser 195 CR1 O His 57 N C CH HC HN CO-O- O Asp 102 E + P 2 Leaving group

24 Chymotrypsin : the substrate binding site Hydrophobic pocket Met192, Gly216, Gly 226 Non-cleavable substrate: N-formyl-L-tryptophan Catalytic site

25 Elastase : the substrate binding site Small amphiphilic binding site Gln192, Val216, Thr226 Non-cleavable substrate : N-formyl-L-alanine Catalytic site

26 Trypsin : the substrate binding site Non-cleavable substrate : N-formyl-L-lysine Catalytic site Hydrophobic pocket Met192, Gly216, Gly 226 Negative charge Asp189

27 1 50 ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN tryp_bovin V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQ el1_pig...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQN thrb_human GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDR klkb_rat.SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPS fa9_human.NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEK fa10_bovin...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTIN tryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLN el1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAG thrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELD klkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMS fa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYN fa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNAN tryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTS el1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQ thrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKG klkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV..GSTCLASG WSSTKPLEWE fa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGR fa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLV tryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVV el1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLH thrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFV klkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLL fa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHV fa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAAN tryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASN el1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASN thrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI... klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKEN fa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE..... fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *

28 Catalytic site conformational flexibility transition state stabilization formation of reaction intermediates defined conformation protein specificity Substrate binding site Summary: protein-substrate interactions ChymotrypsinSer 189Gly 216Gly 226 TrypsinAsp 189 ElastaseVal 216Thr amino acids playing a role in chemical catalysis amino acids not involved in chemical catalysis Catalytic triad (charge relay system)Asp 102,His 57, Ser 195

29 Experimental study of serine protease specificity succinyl-Ala-Ala-Pro-X amino-methylcoumarin specific fluorescent substrates chymotrypsin1, , , , trypsin4,51,80,20,21, trypsin D189S331502,34,716 directed mutagenesis : some trypsin amino acids are replaced by those of chymotrypsin in order to change the enzyme specificity Asp189Ser,site S1 (aa ), loop L1 (aa ), loop L2 (aa ) Tr -> Ch[S1+L1+L2]2, measure the specificity constant k cat /K M enzymes PheTyrTrpLeuLys substrate s

30 Serine protease activation chymotrypsinogen 1245 inactive -chymotrypsin trypsin active -chymotrypsin chymotrypsin active 1 50 ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINEN tryp_bovin V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQ el1_pig...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQN thrb_human GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDR klkb_rat.SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPS fa9_human.NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEK fa10_bovin...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * **

31 - + N Active chymotrypsin

32 - N Inactive chymotrypsine

33 Serine protease inhibition Ala 16 Lys 15 bovine pancreatic trypsin inhibitor SerPins family

34 Cascade of zymogen conversions X VIIaVII Tissue factor Trauma Extrinsic pathway kininogen kallikrein XIIXIIa XIXIa XXa IXIXa VIIIa Intrinsic pathway Cross-linked fibrin network fibrinogen(I) fibrin (Ia) XIIIa prothrombin (II) thrombin (IIa) Va DAMAGED SURFACE CELL DAMAGES CLOTTING

35 Before TPA administration After TPA administration (3h) Anticoagulants Heparin Warfarin Tissue Plasminogen Activator plasminogenplasmin Clot breakdown fibrin fibrin degradation


Download ppt "Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease."

Similar presentations


Ads by Google