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

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 DG‡ = DH‡ - TDS‡ Energy (G) Reaction coordinate B A‡ A DG‡ DG DH‡ : transition state stabilization A‡ DS‡ : 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 :

Intramolecular catalysis and effective concentration Intra-molecular reaction k1 = 0.02 s-1 Vtransfert = k1 [ acyl ] Inter-molecular reaction + k2 = 10-10 M-1s-1 Vtransfert = k2 [ acyl ] [ carboxyl ] Carboxyl effective concentration : k1 / k2 = 2.107 M

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

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 P glucose-6-P + ADP

Michaelis-Menten model of enzyme kinetics Scheme kon koff kcat E + S E.S E + P Chemical processes Conservation equations Steady state [E]0 << [S]0 where Michaelis-Menten equation Hypothesis

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

Graphical representations of Michaelis-Menten equation Eadie-Hofstee V V/[S] Vmax -KM where Michaelis-Menten equation Maximum rate Vmax Michaelis constant KM Vmax KM Vmax/2 Direct representation V [S] 1/V Lineveawer-Burk 1/Vmax -1/KM 1/[S]

The significance of Michaelis-Menten parameters Catalytic constant or turnover kcat : number of substrate molecules processed per enzyme molecule and per second Michaelis constant KM : substrate concentration at which half of the enzymes bind a substrate molecule (and V = Vmax/2) The specificity constant kcat/KM determines the specificity for competing substrates kcat/KM < kon < kdiffusion ≈ 5.108 M-1.s-1 V = [E] [S] kcat/KM For two competing substrates

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

Effect of inhibitors on enzyme kinetics Vmax KM Vmax/2 Direct representation V [S] Eadie-Hofstee plot V V/[S] Vmax -KM Competitive inhibition Vmax unchanged Non-competitive inhibition KM unchanged 1/V Lineveawer-Burk plot 1/Vmax -1/KM 1/[S]

Serine protease family Native substrate C COOH H R1 H2N R2 N O peptide Carboxyl part Amino part Artificial substrate COOH R1 R2 HO O C ester acid alcohol Serine protease specificity Protease R1 Chymotrypsin large hydrophobic amino-acids: Tyr, Trp, Phe, Met Trypsin large positively charged amino-acids: Lys ou Arg, but His Elastase small hydrophobic amino-acids : Ala

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 * * * * ** 51 100 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 **** * * 101 150 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 ** * * * 151 200 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 * * * *** * ****** 201 245 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. * * *

Serine protease : practical study Stopped-flow experiment enzyme substrate Measur-ment chamber C CH3 O HO acetate p-Nitrophenyl acetate C CH3 O NO2 Time (msec) p-Nitrophenol « burst » OH NO2 p-Nitrophenol

Detection of a covalent intermediate ... CH3 O NO2 C CH3 O Chymotrypsin OH NO2 Chymotrypsin + + C CH3 O HO E + S E.S E-P2 E Time (msec) p-Nitrophenol « burst » P1 P2 Energy (G) E-P2 + P1 E + S E + P1 + P2 Reaction coordinate

di-isopropyl-phosphofluoridate … bound to serine 195 Irreversible inhibition of serine proteases by DIPF C CH3 H O H3C P F C CH3 H O H3C P CH2 OH Chymotrypsin 195 + CH2 O Chymotrypsin 195 + HF di-isopropyl-phosphofluoridate

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 * * * * ** 51 100 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 **** * * 101 150 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 ** * * * 151 200 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 * * * *** * ****** 201 245 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. * * *

Histidine 57 is also part of serine protease catalytic site Irreversible inhibition of chymotrypsin byTPCK Specificity group Reactive group H O Chymotrypsin + CH2 C C CH2Cl Chymotrypsin 57 NH 57 CH2 CH2 C O S O C + HN ClCH2 N CH CH HC NH HC N tosyl-L-phenylalanine chloromethyl ketone

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 * * * * ** 51 100 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 **** * * 101 150 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 ** * * * 151 200 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 * * * *** * ****** 201 245 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. * * *

“catalytic triad” or “charge relay system” The hydrogen bond network at the serine protease catalytic site: Serine 195, Histidine 57 and Aspartate 102 His 57 N C CH HC HN C O- O Asp 102 CH2 HO Ser 195 NH C CH HC + HN His 57 C O- O Asp 102 CH2 -O Ser 195 “catalytic triad” or “charge relay system” NH C CH HC N His 57 C OH O Asp 102 CH2 -O Ser 195

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 * * * * ** 51 100 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 **** * * 101 150 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 ** * * * 151 200 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 * * * *** * ****** 201 245 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. * * *

Serine protease mechanisms NH C CH HC + HN His 57 CH2 O Ser 195 O- Asp 102 R2 N R1 H Transition state E.S Substrate His 57 N C CH HC HN CH2 HO Ser 195 O- O Asp 102 R2 R1 H E + S Covalent intermediate CH2 O Ser 195 R2 N C R1 H His 57 CH HC HN O- Asp 102 E-P2 + P1 Leaving group

Serine protease mechanisms Transition state NH C CH HC + HN His 57 CH2 O Ser 195 O- Asp 102 R1 H E.P2 Water molecule activation CH2 O Ser 195 C R1 H His 57 N CH HC HN O- Asp 102 E-P2 CH2 HO Ser 195 C R1 O His 57 N CH HC HN O- Asp 102 E + P2 Leaving group

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

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

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

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 * * * * ** 51 100 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 **** * * 101 150 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 ** * * * 151 200 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 * * * *** * ****** 201 245 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. * * *

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

Experimental study of serine protease specificity succinyl-Ala-Ala-Pro-X amino-methylcoumarin specific fluorescent substrates directed mutagenesis : some trypsin amino acids are replaced by those of chymotrypsin in order to change the enzyme specificity Asp189Ser,site S1 (aa 189-195), loop L1 (aa 214-220), loop L2 (aa 225-228) measure the specificity constant kcat/KM enzymes Phe Tyr Trp Leu Lys substrates chymotrypsin 1,6.106 4,5.106 6,8.106 1,2.105 850 trypsin 4,5 1,8 0,2 0,2 1,2.106 trypsin D189S 33 150 2,3 4,7 16 Tr -> Ch[S1+L1+L2] 2,8.103 2.104 2.103 103 34

Serine protease activation chymotrypsinogen 1 245 inactive p-chymotrypsin 245 15 16 1 trypsin active a-chymotrypsin 245 16 13 146 149 1 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 * * * * **

N + - Active chymotrypsin

Inactive chymotrypsine - N Inactive chymotrypsine

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

Cascade of zymogen conversions kininogen kallikrein XII XIIa XI XIa X Xa IX IXa VIIIa Intrinsic pathway X VIIa VII Tissue factor Trauma Extrinsic pathway CELL DAMAGES DAMAGED SURFACE Cross-linked fibrin network fibrinogen(I) fibrin (Ia) XIIIa prothrombin (II) thrombin (IIa) Va CLOTTING

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