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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 G‡ = H‡ - TS‡
kA→ B =kTh
exp −ΔG‡
RT
⎛
⎝ ⎜ ⎜
⎞
⎠ ⎟ ⎟
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 :
Intramolecular catalysis and effective concentration
O
CO2H
CH2
OC
C
O
O
O
CO2H
C
CH2
OCO
OIntra-molecular reaction
k1 = 0.02 s-1
Vtransfert = k1 [ acyl ]
O
CH2
OC
C
O
O
C
CH2
OCO
O
O
Inter-molecular reaction
+ +
k2 = 10-10 M-1s-1Vtransfert = k2 [ acyl ] [ carboxyl ]
Carboxyl effective concentration : k1 / k2 = 2.107 M
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
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
Hypothesis
Michaelis-Menten model of enzyme kinetics
Schemekon
koff
kcat
E + S E.S E + P
V =Vmax
S[ ]totS[ ]tot +KM
Vmax =kcat E[ ]tot KM =koff +kcat
kon
where
Michaelis-Menten equation
d E.S[ ]dt
=kon E[ ].S[ ] −koff E.S[ ] −kcat E.S[ ]
V =d P[ ]dt
=kcat E.S[ ]
E[ ]tot = E[ ]+ E.S[ ]
Chemical processes
€
S[ ]tot=S[ ]+E.S[ ]+P[ ]Conservation equations
Steady state
[E]0 << [S]0
How enzymes are studied ?
colored or fluorescent substrates or products : spectrophotometric methods
radioactive substrates and products : filtration methods
reaction coupling
Kinetic methods
time
Pro
duc
t co
ncen
trat
ion
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
V =Vmax
S[ ]totS[ ]tot +KM
Vmax =kcat E[ ]tot KM =koff +kcat
kon
where
Michaelis-Menten equation
Maximum rate
VmaxMichaelis constant
KM
Vmax
KM
Vmax/2
Direct representationV
[S]
Eadie-HofsteeV
V/[S]
Vmax
-KM
1/VLineveawer-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
VAVB
=kcat KM( )A A[ ]
kcat KM( )B B[ ]
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 representationV
[S]
Eadie-Hofstee plotV
V/[S]
Vmax
-KM
1/VLineveawer-Burk plot
1/Vmax
-1/KM
1/[S]
Competitive inhibitionVmax unchanged
Non-competitive inhibitionKM unchanged
Serine protease family
Artificial substrate
COOHR1 R2HOR2OCR1
O
ester acid alcohol
Native substrate
C COOH
H
R1
H2N C COOH
H
R2
H2NC COOH
H
R2
NC C
H
R1
H2N
O
H
peptide Carboxyl part Amino part
Serine protease specificity
Protease R1Chymotrypsin large hydrophobic amino-acids: Tyr, Trp, Phe, MetTrypsin large positively charged amino-acids: Lys ou Arg, but HisElastase small hydrophobic amino-acids : Ala
1 50ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINENtryp_bovin .........V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQel1_pig ...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQNthrb_human .......... ...GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDRklkb_rat .SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPSfa9_human .NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEKfa10_bovin ...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** 51 100ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTINtryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLNel1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAGthrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELDklkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMSfa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYNfa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * 101 150ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNANtryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTSel1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQthrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKGklkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV ..GSTCLASG WSSTKPLEWEfa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGRfa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * 151 200ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLVtryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVVel1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLHthrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFVklkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLLfa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHVfa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** 201 245ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAANtryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASNel1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASNthrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI...klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKENfa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE .....fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *
Serine protease : practical study
Stopped-flow experiment
enzyme
substrate
Measur-ment
chamber
Time (msec)
p-N
itrop
heno
l
« burst »
p-Nitrophenyl acetate
C CH3
O
O
NO2
p-Nitrophenol
OH
NO2
C CH3
O
HO
acetate
Detection of a covalent intermediate ...
Time (msec)p-
Nitr
ophe
nol
« burst »
C CH3
O
O
NO2
OH
NO2
E + S
Chymotrypsin + +
C CH3
O
HO
C CH3
O
OChymotrypsin
E.S E-P2 E
P1 P2
Ene
rgy
(G)
Reaction coordinate
E + P1 + P2
E + S
E-P2 + P1
… bound to serine 195
Irreversible inhibition of serine proteases by DIPF
+CH2 OHChymotrypsin195
C CH3
H
O
H3C
P OF
C CH3
H
O
H3C
+ HF
C CH3
H
O
H3C
P O
C CH3
H
O
H3C
CH2 OChymotrypsin195
di-isopropyl-phosphofluoridate
1 50ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINENtryp_bovin .........V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQel1_pig ...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQNthrb_human .......... ...GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDRklkb_rat .SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPSfa9_human .NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEKfa10_bovin ...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** 51 100ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTINtryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLNel1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAGthrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELDklkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMSfa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYNfa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * 101 150ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNANtryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTSel1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQthrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKGklkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV ..GSTCLASG WSSTKPLEWEfa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGRfa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * 151 200ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLVtryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVVel1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLHthrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFVklkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLLfa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHVfa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** 201 245ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAANtryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASNel1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASNthrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI...klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKENfa9_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
+Chymotrypsin
57
tosyl-L-phenylalanine chloromethyl
ketone
NH
CH2
C
CHHC
+ HN
C C
H
CH2
NH
S OO
O
CH2Cl Chymotrypsin
57CH2
ClCH2
N
C
CHHC
N
Specificity group
Reactive group
1 50ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINENtryp_bovin .........V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQel1_pig ...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQNthrb_human .......... ...GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDRklkb_rat .SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPSfa9_human .NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEKfa10_bovin ...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** 51 100ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTINtryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLNel1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAGthrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELDklkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMSfa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYNfa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * 101 150ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNANtryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTSel1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQthrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKGklkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV ..GSTCLASG WSSTKPLEWEfa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGRfa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * 151 200ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLVtryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVVel1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLHthrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFVklkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLLfa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHVfa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** 201 245ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAANtryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASNel1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASNthrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI...klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKENfa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE .....fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *
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
CHHC
HN
NH
C
CHHC
+ HN
His 57
CH2HO Ser 195
C O-
O
Asp 102
CH2-O Ser 195
C O-
O
Asp 102
NH
C
CHHC
N
His 57
CH2-O Ser 195
C OH
O
Asp 102
1 50ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINENtryp_bovin .........V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQel1_pig ...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQNthrb_human .......... ...GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDRklkb_rat .SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPSfa9_human .NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEKfa10_bovin ...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** 51 100ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTINtryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLNel1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAGthrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELDklkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMSfa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYNfa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * 101 150ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNANtryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTSel1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQthrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKGklkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV ..GSTCLASG WSSTKPLEWEfa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGRfa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * 151 200ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLVtryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVVel1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLHthrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFVklkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLLfa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHVfa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** 201 245ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAANtryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASNel1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASNthrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI...klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKENfa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE .....fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *
Serine protease mechanisms
Substrate
His 57
N
C
CHHC
HN
CH2HO
Ser 195C O-
O
Asp 102
R2 N C R1
O
H
E + S
NH
C
CHHC
+ HN
His 57
CH2O
Ser 195
C O-
O
Asp 102
R2 N C R1
O-H
Transition stateE.S
Covalent intermediate
CH2O
Ser 195
R2 N C R1
H O
His 57
N
C
CH
HC
HNC O-
O
Asp 102
H
E-P2 + P1
Leaving group
Serine protease mechanisms
Water molecule activation
CH2O
Ser 195
O C R1
H O
His 57
N
C
CH
HC
HNC O-
O
Asp 102
H
E-P2
Transition state
NH
C
CHHC
+ HN
His 57
CH2O
Ser 195
C O-
O
Asp 102
O C R1
O-H
E.P2
CH2HO
Ser 195
C R1
O
His 57
N
C
CH
HC
HNC O-
O
Asp 102
E + P2
Leaving group
Chymotrypsin : the substrate binding site
Hydrophobic pocket
Met192, Gly216, Gly 226
Non-cleavable substrate:N-formyl-L-tryptophan
Catalytic site
Elastase : the substrate binding site
Small amphiphilic binding site
Gln192, Val216, Thr226
Non-cleavable substrate :N-formyl-L-alanine
Catalytic site
Trypsin : the substrate binding site
Non-cleavable substrate :N-formyl-L-lysine
Catalytic site
Hydrophobic pocket
Met192, Gly216, Gly 226
Negative chargeAsp189
1 50ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINENtryp_bovin .........V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQel1_pig ...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQNthrb_human .......... ...GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDRklkb_rat .SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPSfa9_human .NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEKfa10_bovin ...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF * * * * ** 51 100ctra_bovin WVVTAAHCGV TTSDVVVAGE FDQGSSSEKI QKLKIAKVFK NSKYNSLTINtryp_bovin WVVSAAHCYK SGI.QVRLGE DNINVVEGNE QFISASKSIV HPSYNSNTLNel1_pig WVMTAAHCVD RETFRVVVGE HNLNQNDGTE QYVGVQKIVV HPYWNTVAAGthrb_human WVLTAAHCLL YPOLLVRIGK HSRTRYERNI EKIMLEKIYI HPRYNWRELDklkb_rat WVITAAHC.Y SHNYHVLLGR NNLFKDEPFA QYRVVNQSFP HPDYNPFFMSfa9_human WIVTAAHCVE TGVKTVVAGE HNIEETEHTE QKRNVIRIIP HHNYNAAIYNfa10_bovin YVLTAAHCLH QARFTVRVGD RNTEQEEGNE MAHEVEMTVK HSRFVKETYD **** * * 101 150ctra_bovin NDITLLKLST AASFSQTVSA VCLPSASDDF AAGTTCVTTG WGLTRYTNANtryp_bovin NDIMLIKLKS AASLNSRVAS ISLPTSCA.. SAGTQCLISG WGNTKSSGTSel1_pig YDIALLRLAQ SVTLNSYVQL GVLPRAGTIL ANNSPCYITG WGLTR.TNGQthrb_human RDIALMKLKK PVAFSDYIHP VCLPDAASLL QAGYKGRVTG WGNLKETGKGklkb_rat NDLMLLHLSE PADITDGVKV IDLPTEEPKV ..GSTCLASG WSSTKPLEWEfa9_human HDIALLELDE PLVLNSYVTP ICIADKTNIF LKFGSGYVSG WGRV.FHKGRfa10_bovin FDIAVLRLKT PIRFRRNVAP ACLPEAEATL MTQKTGIVSG FGRTH.EKGR ** * * * 151 200ctra_bovin TPDRLQQASL PLLSNTNCKK YWGTKIKDAM ICAGASGVSS CMGDSGGPLVtryp_bovin YPDVLKCLKA PILSDSSCKS AYPGQITSNM FCAGYGGKDS CQGDSGGPVVel1_pig LAQTLQQAYL PTVDYAICSS YWGSTVKNSM VCAGGDGVSG CQGDSGGPLHthrb_human QPSVLQVVNL PIVERPVCKD STRIRITDNM FCAGYKRGDA CEGDSGGPFVklkb_rat FPDDLQCVNI NILSNEKCIK AHTQMVTDVM LCAGEGGKDT CNGDSGGPLLfa9_human SALVLQYLRV PLVDRATCLR STKFTIYNNM FCAGFGGRDS CQGDSGGPHVfa10_bovin LSSTLKMLEV PYVDRSTCKL SSSFTITPNM FCAGYQPEDA CQGDSGGPHV * * * *** * ****** 201 245ctra_bovin CKKNGAWTLV GIVSWGSSTC STSTPGVYAR VTALVNWVQQ TLAANtryp_bovin CSGK....LQ GIVSWGSGCA QKNKPGVYTK VCNYVSWIKQ TIASNel1_pig CLVNGQYAVH GVTSFVSRLG CTRKPTVFTR VSAYISWINN VIASNthrb_human MKSNNRWYQM GIVSWGEGCD RDGKYGFYTH VFRLKKWIQK VI...klkb_rat CDG....VLQ GITSWSSVPC GTNRPAIYTK LIKFTSWIKE VMKENfa9_human TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE .....fa10_bovin TRFKDTYFVT GIVSWGEGCA RKGKFGVYTK VSNFLKWIDK IMKA. * * *
Catalytic site
conformational flexibility
transition state stabilization
formation of reaction intermediates
defined conformation
protein specificity
Substrate binding site
Summary: protein-substrate interactions
Chymotrypsin Ser 189 Gly 216 Gly 226
Trypsin Asp 189
Elastase Val 216 Thr 226-
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
Experimental study of serine protease specificity
succinyl-Ala-Ala-Pro-X amino-methylcoumarin
specific fluorescent 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
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)
Tr -> Ch[S1+L1+L2] 2,8.103 2.104 2.103 103 34
measure the specificity constant kcat/KM
enzymes Phe Tyr Trp Leu Lys substrates
Serine protease activation
chymotrypsinogen 1 245 inactive
-chymotrypsin 24515 161
trypsin
active
-chymotrypsin 2451613 146 1491
chymotrypsinchymotrypsin
active
1 50ctra_bovin CGVPAIQPVL SGLSRIVNGE EAVPGSWPWQ VSLQDKTGFH FCGGSLINENtryp_bovin .........V DDDDKIVGGY TCGANTVPYQ VSLN..SGYH FCGGSLINSQel1_pig ...HSTQDFP ETNARVVGGT EAQRNSWPSQ ISLQYRSGSH TCGGTLIRQNthrb_human .......... ...GRIVEGS DAEIGMSPWQ VMLFRKSPEL LCGASLISDRklkb_rat .SVGRIDAAP PGQSRVVGGY KCEKNSQPWQ VAVINR...Y LCGGVLIDPSfa9_human .NITQSTQSF NDFTRVVGGE DAKPGQFPWQ VVLNGKVD.A FCGGSIVNEKfa10_bovin ...PSAGEDG SQVVRIVGGR DCAEGECPWQ ALLVNEENEG FCGGTILNEF
* * * * **
- +
N
Active chymotrypsin
-
N
Inactive chymotrypsine
Serine protease inhibition
Ala 16Lys 15
bovine pancreatic trypsin inhibitor
SerPins family
Cascade of zymogen conversions
X
VIIa VII
Tissue factor
Trauma
Extrinsic pathwaykininogenkallikrein
XII XIIa
XI XIa
X Xa
IX IXaVIIIa
Intrinsic pathway
Cross-linked fibrin network
fibrinogen(I) fibrin(Ia)
XIIIa
prothrombin(II)
thrombin(IIa)
Va
DAMAGED SURFACE
CELL DAMAGES
CLOTTING
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Before TPA administration
After TPA administration (3h)
Anticoagulants
Heparin
Warfarin
Tissue Plasminogen Activator
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plasminogen plasmin
Clot breakdown
fibrin fibrin degradation
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