Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics...

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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

Transcript of Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics...

Page 1: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 2: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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 :

Page 3: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 4: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 5: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 6: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 7: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 8: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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]

Page 9: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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[ ]

Page 10: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 11: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 12: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 13: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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. * * *

Page 14: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 15: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 16: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

… 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

Page 17: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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. * * *

Page 18: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 19: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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. * * *

Page 20: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 21: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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. * * *

Page 22: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 23: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 24: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

Chymotrypsin : the substrate binding site

Hydrophobic pocket

Met192, Gly216, Gly 226

Non-cleavable substrate:N-formyl-L-tryptophan

Catalytic site

Page 25: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

Elastase : the substrate binding site

Small amphiphilic binding site

Gln192, Val216, Thr226

Non-cleavable substrate :N-formyl-L-alanine

Catalytic site

Page 26: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

Trypsin : the substrate binding site

Non-cleavable substrate :N-formyl-L-lysine

Catalytic site

Hydrophobic pocket

Met192, Gly216, Gly 226

Negative chargeAsp189

Page 27: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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. * * *

Page 28: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 29: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 30: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

* * * * **

Page 31: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

- +

N

Active chymotrypsin

Page 32: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

-

N

Inactive chymotrypsine

Page 33: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

Serine protease inhibition

Ala 16Lys 15

bovine pancreatic trypsin inhibitor

SerPins family

Page 34: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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

Page 35: Enzyme catalysis Basic concepts in chemical catalysis The Michaelis-Menten model of enzyme kinetics Structure-function relationship: the serine protease.

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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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