Fig. S2. Examples of fitting of steady-state kinetics to fluorescent fractions of α-elastin. Progress curves (25 ºC in TNC buffer) fitted with apparent steady-state enzyme kinetics parameters are illustrated for MMP-12digestion of fEln-100 (a),fEln-20 (b), or MMP-12(I180S) digestion of fEln-100 (c).
Fig. S3. Examples of fitting of catalytic efficiencyof MMP-12 digestion of DQ-collagens. The progress curves (25 ºC in TNC buffer)were measured by exciting at 490 nm and detecting at 512 nm using an SLM-Aminco 8100 upgraded by ISS. The progress curves werefitted to apparent kcat/Km(see Palmier and Van Doren, 2007,Anal. Biochem.) for DQ-collagen IV (a,c) and DQ-collagen I (b,d) for WT MMP-12 (a,b) and MMP-12(I180S). Since the mass of DQ-collagens was undefined (ran on SDS-PAGE as a smear of bands from high to low MW), individual kcatandKm could not be determined.kcat/Kmwas fitted on the basis of [MMP-12] at low [S],per the Michaelis-Menten equation.
Fig. S4. Consensus sequences representing 286 matrix metalloproteinases divided into 54 subfamilies with > 87% sequence identity in each. In the summary line, a capital “X” marks a class-specific residue conserved within ≥17 of 18 groups. A lower case “x” marks a nearly class specific site conserved within ≥15 of 18 groups. Since the fibronectin-like inserts in MMP-2 and -9 disrupted alignments with catalytic domains at HSSP and ET Viewer servers, MMP-2 and -9 sequences are omitted.
GRP1 GSRWKV.....K-LTYRISK...... YP-RL-R--VDKEIAK.AF-VWS.EYTDL-FT-KK-...... --VHIDIRFE-N...... EHGDG...... DPFDGPG
GRP2 GSRWRV.....KNLTYK-SK...... YPKRLKRVDVDAEIGR.AFAVWS.EDTDLTFT-KT-...... GPVHIEIKFVES...... EHGDG...... DAFDGQG
GRP3 Hatch enz GIAWPR.....NVAVTYSFG.T....L-NDL-Q-AIKNE-RR.AFQVW-.DVS-LTFREVVDS...... SSVDIRIKFGSY...... EHGDG...... ISFDGQG
GRP4 Hatch enz .ITWSR.....NQPVTYSFG.A....LTSDLNQND-KDEIRR.AFRVWD.DVSGLSFREVPDT...... TSVDIRIKFGSY...... DHGDG...... ISFDGRG
GRP5 MMP11-like------.....-----RILRFP...... WQL--EQVRQT-AE.AL-VWS.-VTPLTFTEVHE...... G-ADI-IDF-RY...... W-GD-...... LPFDGPG
GRP6 MMP11-likeGGRWDK.....T-LTYKIIRFP...... WQL-K-KVRRTI-E.ALKVWS.-VTPLTFTEV-E...... GR-DI-IDFTRY...... WHGDN...... LPFDGPG
GRP7 MMP3,10 -PKW-KTH.....LTYR...... IVNYT-DLP-DA-D-A-EK.A-KVWE.EVTPLTFSR-YEGE...... ADIMISF-VR...... EHGD...... F-PFDGPG
GRP8 MMP10 -PKWRKTH.....LTYR...... IVN-T-DLP-DAVDSAIEK.A-KVWE.EVTPLTFSRLYEGE...... ADIMISFAVK...... EHGD...... FYSFDG-G
GRP9 MMP10 SPKWRK-H.....I-YR...... IVNYT-DLPR-SVDSAIE-.ALKVWE.EVTPLTFSRISEGE...... ADIMISFAVG...... EHGD...... FYPFDG-G
GRP10 MMP3 SPKWRK-H.....I-YR...... IVNYT-DLPR-SVDSAIE-.ALKVWE.EVTPLTFSRISEGE...... ADIMISFAV-...... EHGD...... F-PFDGPG
GRP11 MMP7 NP-W-K--.....LTYS...... IVNYTPDL-RQVVD-AIQR.AFGVWS.NVTPLQFTKVSSG-...... ADI-IRFGAR...... THGDS.....-.PFDGPS
GRP12 MMP8,18 R-RW-KTQ.....L-YR...... IVNYTPDLPR--VDEAIR-.AFKVWS.DVTPLKFTR-SSRR...... ADI-IQFGAR...... SHGD-.....I.PFDGPN
GRP13 MMP8,18 SSAWQK-D.....LTYR...... I-NFTPDLPQADVE-AIQR.AFKVWS.DVTPLTFTRIYNEV...... SDIEISF-AG...... DHKDN.....S.PFDG-G
GRP14 MMP3/10a RPTWRTTS.....LTYR...... IL-YTPDMAEADVDTAIRR.AFKVWS.DVTPLTFSRIYEGT...... ADIQISFGAG...... VHGDF.....Y.PFDGPH
GRP15 MMP3/10b RP-WRTHA.....LTYR...... ILNYTPDMARADVDTAIQK.AFKVWS.DVTPLTFT-IYYGT...... ADIQISF-AR...... EHGDF.....-.PFDGP-
GRP16 MMP1-likeNPVW-KKD.....LTYR...... I-NYTPDMTR-EVDRAIQK.AFKVWS.DVTPLTFTRVNDGV...... ADIEISFAA-...... -H-DF.....Y.PFDGPY
GRP17 MMP13 TLKW---N.....LTYR...... IVNYTPD--HSEVEKAF-K.AFKVWS.DVTPLNFTR---G-...... ADIMISFG-K...... EHGD...... FYPFDGPS
GRP18 MMP13 KLKWPRNN.....LTYR...... IVNYTPDLSTS-VDRAIKK.ALKVWS.DVTPLNFTRLRTGT...... ADIMV-FGKK...... EHGD...... YYPFDGPD
GRP19 MMP13 -LKW---N.....-TYR...... IENYTPDMTHAEVDRAIKK.AFRVWS.EVTPLNFTRLRSGT...... ADIMISFG--...... EHGD...... FYPFDGP-
GRP20 MMP13 DYKWKKHQ.....LTYR...... I-NYTPDMSVAEVDDSISK.ALKVWA.DVTPLRFTRIYSGT...... ADIMISFATG...... DHRD...... GYPFDGPN
GRP21 MMP10 NPKW-KTT.....-TYR...... ILNYTPDITKSEVDYAIAQ.AFRVWS.DVTPLNFQKLNS-D...... ADI-ISFNT-...... AHGD...... F-SFDGPN
GRP22 MMP8 NPKWE-TN.....LTY-...... I-NYTPQLSE--VE-AIK-.AFE-WS.-ASPL-FTRISQG-...... ADINIAF-QR...... DHGD...... NSPFDGPN
GRP23 MMP8 SPKWTHTN.....LTY-...... IINHTPQ-S-AEVKT-IEK.AF--WS.V-S-LTFTE-L-GE...... ADINIAFVSR...... DHGD...... NSPFDGPN
GRP24 MMP1 ---WE-T-.....L-YR...... IENYTPDL-R--VD--I-K.AFQLWS.NV-PLTFTKVS-GQ...... ADIMISFVRG...... DHRD...... NSPFDGP-
GRP25 MMP27 LPGWRKYN.....LTYR...... I-NYTPDMAR--VD-AIQ-.-LEVWS.-VTPL-FTKI-KGI...... ADIMIAFRTR...... VHG-C.....PRYFDGPL
GRP26 MMP27 LPGWRKY-.....LTYR...... IMNYTPDMT-ADVDEAIQK.ALQVWS.KVTPL-FTRISKGV...... ADIMIAFRT------VHGWC.....PRHFDGPL
GRP27MMP12 G--WRKHY.....ITYR...... INNYTPDMNR-DVDYAI-K.AFQVWS.NVTPLKFS-INTG-...... ADILVVFARG...... AHGD...... FHAFDGKG
GRP28 MMP12 RPVWRKHY.....ITYR...... IKNYTPDMKREDVEYAIQK.AFQVWS.DVTPLKFRKITTG-...... ADIMILFASG...... AHGD...... YGAFDGRG
GRP29 MMP12 RSRWMKRY.....LTYR...... IYNYTPDMKR-DVDYIFQK.AFQVWS.DVTPLRFRK-HK-E...... ADI-ILFAFG...... --GD...... F--FDGKG
GRP30 MMP20 EPKWKKN-.....LTYR...... -SKYT-SM---EV--A--M.AL-AW-.-AVPL-F-R-N-GE...... ADIMISFE-G...... DHGD...... SYPFDGPR
GRP31 MMP7 SPKWTSKV.....VTYR...... IVSYTRDLPH-TVDRLVSK.ALNMWG.KEIPLHFRKVV-GT...... ADIMIGFARG...... AHGD...... -YPFDGPG
GRP32 MMP7 SPKWHSR-.....VTYR...... IVSYT-DLPR--VDQIVK-.ALRMWS.MQIPLNFKRVSWGT...... ADIIIGFAR-...... DHGD...... -FPFDGPG
GRP33 KPRWKQSV.....VTYR...... ITEY-TQLSQREVDSTIAK.AFQLYS.DVIPVDFKQIFSGT...... ADIMILFKGG...... -HGD...... FYPFDGPN
GRP34 MT1MMP GLKWQ....-NE.ITFC...... IQNYTP-VGEYAT-EAIRK.AFRVWE.SATPLRFREV-YAYIR-G...-EKQ....AD--I-F-EG...... -HGD...... -T-FDGEG
GRP35 MT1MMP GLKWQ....HKD.ITFC...... IQNYTPKIGEYST-EAIRR.AFKVWE.SVTPLRF-EVRYVDI-DG...YTKH....ADIMLFFAEG...... FHGD...... STPFDGEG
GRP36 MTMMP -LKW-....K--.-TFS...... IQNYTPKVGEYET-EAIR-.AFK-WE.-VTPLRFREIPYSYIRDK...VE-F....ADIMIFF-EG...... FHGD...... SSPFDGEG
GRP37 MTMMP -L---....---.----...... IQNYTPKVGEYETFEAIRK.AFKVWE.SVTPLRFREISYSDIRDK...VVDF....ADIMLFFADG...... FHGD...... ASPFDGEG
GRP38 MTMMP,28 GHKW-....K--.-T-S...... IQNYTPKIGEY-S-EAIRR.AF-VWE.-VTPLTFDEIP--E-KYG...RRKE....PDIMIFFASG...... FHGD...... SSPFDGEG
GRP39 MT5MMP GQKWR....-K-.ITYS...... -HN-TPKVGE-DT--AIRQ.AFDVWQ.-VTPL-F-EV-Y-EIK--....-KE....ADIMIFFASG...... FHGD...... SSPFDGEG
GRP40 MT3MMP GQKW-....--H.I-YS...... -KN-TPKVG---T--AIRR.AFDVWQ.-VTPL-FE--PY--L---....--D....VDITI-FASG...... FHGD...... -SPFDGEG
GRP41 MT2MMP G--W-....N-H.LTFS...... IQNYTEKLGWY-S-EAVRR.AF-VWE.Q-TPLVFQEV-Y-DIRLR...R--E....ADIMVLFASG...... FHGD...... SSPFDG-G
GRP42 MMP19 LG-WRK.....KHLTFRI-NLP...... STLP--TARAAL-Q.AFQ-WS.--APLTF-EV-A...... G-ADIRLSFHGR...... -S-.....YCSN-FDGPG
GRP43 LGHWRK.....K-LTFRI-NVP...... STLS--RVRAALHQ.AFKYWS.-VAPLTFREVKA...... GWADIRLSFHGR...... QS-.....YCSN-FDGPG
GRP44 MMP26 RCKWNK-T.....LTYR...... IINYPHDMKPS-VKDSIYN.AVSIWS.NVTPLIFQQVQN-D...... ADIK-SFWQW...... AHED...... GWPFDGPG
GRP45 MMP28 G-KWYK.....QHLSYRLVNWP...... --LPEPAVRGAVRA.AFQLWS.NVSALEFWEAPAT...... -PADIRLTFFQG...... DHN....DGL-NAFDGPG
GRP46 MT6MMP GSVWKKRT.....LTWRVRSFP...QSSQ.LSQETVRVLM-.YALMAWGM.ES-LTFHEVDS-...... QGQEPDILIDFARA...... FHQDS...... YPFDGLG
GRP47 MT6MMP GS-W-KRT.....LTWRV-SFP...Q-S-.L-QETVRTLMH.HALTTWG-.------...... -PTEPDILIDFARA...... YHQDS...... YPFDGQG
GRP48 MT6MMP GSVWKKRT.....LTWSI-SFS...Q-S-.LS---VRTL-S.YAL-VWA-.-SGLTFQEV-S...... QYQEPDI-IHF-RA...... YHQDS...... YPFDG-G
GRP49 MT4MMP -T-W-KR-.....LSWRVRTFP...RDSP.LG-DTVRALM-.YALKVWSD.ITPLNFHEVAG...... --ADIQIDFS-A...... DHND-...... YPFDGPG
GRP50 MT4MMP --KWNKR-.....LSWRVRTFP...KES--LG-DTVRALMY.YALKVWSD.I-PLNFHEVAG...... N-ADIQIDF-KA...... DHNDG...... YPFDGPG
GRP51 QIKWKKRH.....LSWRVRSFP...KDSPSLGRDTVRALMH.YALKVWSD.IAPLDFHEVAG...... SDADIQIDFTKA...... DHRDG...... YPFDGPG
GRP52 MMP2-like------.....----.....MVNRSMP..DASKVERMVQT.ALDVWA.NHSKLTFREVYSDQ...... ADIQILFARR...... AHGDG...... YKFDGPG
GRP53 QFKW-K.....-RLTYSIESWS...... SDL-K-DVRRAI-E.AYGLWS.KVTPLEFSEVPAG...... STSDIKIRFGV...... RNHN.....DPWPFDGEG
GRP54 SESWRKR-.....ITYF...... IANWSSKVGED-VAKFM--.AF-EW-.KYS-L-FVRVYDPS...... ADIIVGFGSG...... HHGDN...... YPFDGPG
SUMMARY xxw xxxx x xX XxxXxx aXxxWX xXxlxfxxxxx XdixXXFxxx xXX xFDGxX
Fig. S4 continued
GRP1 GTLAHAYFPVYGG...... DAHFDDAE-WTI-K...... -RGTNLFQVAAHEFGHSLGLSHSDVRSALMAPFYRGYDP...... VFRLDSDD-QGIQ-LYG
GRP2 MMP1 GTLAHAFFPVFGG...... DAHFDDAELWTIGS...... PRGTNLFQVAAHEFGHSLGLSHSDQSSALMAPFYRGFEP...... VF-LD-DDKAAIQSLYG
GRP3 Hatch enz GVLAHAFLPRNG...... DAHFDDSE-WTIGT...... N-GTNLFQVAAHEFGHSLGLYHSDVQ-ALMYPYYRGYNP...... NFNLDRDDIAGITSLYG
GRP4 Hatch enz GVLAHAFLPRNG...... DAHFDDSETWT-GT...... RSGTNLFQVAAHEFGHSLGLYHSTVRSALMYPYYQGYVP...... NFRLD-DDIAGIRSLYG
GRP5 MMP11-likeGILAH-F-PKTHR.....EGDVHFDYDETWTIG--...... -GTDLLQVAAHEFGH-LGLQ-TTAAKALMS-FYTFRYP...... LSLSPDD-RG-Q-LYG
GRP6 MMP11-likeGILAHAFFPKTHR.....EGDVHFDYDE-WTIGNN...... -GTDLLQVAAHEFGH-LGLQH---SKSLMSPFY-FRYP...... LSLS-DDK-GIQ-LYG
GRP7 MMP3,10 NVLAHAYAPG.....PGINGDAHFDDDEQWTKDT...... TG-NLFLVAAHE-GHSLGLFHSAN-EALMYP-Y---T....DL-RFRLSQDD--GIQSLYG
GRP8 MMP10 HSLAHAYPPG.....PGLYGDIHFDDDE-WTEDA...... SGTNLF-VAAHELGHSLGLFHSANTEALMYPLYNSFT....EL--F-LSQDDVNGIQSLYG
GRP9 MMP10 QSLAHAYPPG.....PGFYGD-HFDDDEKW-L-P...... SGTNLFLVAAHELGHSLGLFHS--KESLMYPVYRFST....S-AN--LSQDDIEGIQSLYG
GRP10 MMP3 -VLAHAYAPG.....PG-NGDAHFDDDER-T-DV...... TGTNLFLVAAHELGHSLGL-HSA-AEALMYPVYKSST....DL-RFHLSQDDVDGIQSLYG
GRP11 MMP7 GVLAHAYGPG.....RGIGGDAHFDEDERWTSSR-...... GFNLFLVAAHEFGHSLGLDHSTDP-ALMFPTYHYV.....-T--FRLSQDDINGIQSI--
GRP12 MMP8,18 GVLAHA-APG.....-GIGGDAHFDEDERWTS-SA...... GFNLFLVAAHEFGHSLGLDHSRD-RALMFP-YRY-.....NTRNFRLPQDDVNGIQSIYG
GRP13 MMP8,18 GILAHAFQPG.....NGIGGDAHFDEDE-WTKTS-...... -YNLFLVAAHEFGHSLGLSHSTD-GALMYP-YS-T.....DP--FQLPQDDINAIQYLYG
GRP14 MMP3/10a GTLAHAFAPG.....NSIGGDAHFDEDETWTAGSA...... GYNLFLVAAHEFGHSLGL-HSGDRSALMYPTYSY-.....DPARFRLPQDDVDGIQALYG
GRP15 MMP3/10b GTLAHAFAP-.....TGIGGDAHFDEDE-W-K-S-...... GTNLFLVAAHEFGHSLGLSHSNDR-ALMFPTYSYT.....DPAR--LPKDDINGIQA-YG
GRP16 MMP1-likeGTLAHAFAPG.....NN-GGDAHFDEDEDWTSGSV...... GFNLFLVAAHEFGHSLGL-HSNDP-ALMYP-YHYV.....-P-T-QLPQDD-NGIQSLYG
GRP17 MMP13 G-LAHAFPPG.....PN-GGDAHF-DDETWTSSSK...... GYNLF-VAAHE-GHSLGLDHSKDPGALMFPIYTYT.....GK------
GRP18 MMP13 GLLAHAFPPG.....EK-GGDTHFDDDEMFSTDNK...... GYNLFVVAAHEFGHALGLDHSRDPGSLMFPVYTYT.....ETSRFVLPDDDVQGIQ-LYG
GRP19 MMP13 GLLAHAFPPG.....--IGGDTHFDDDETFTSGS-...... GYNLFIVAAHEFGHALGLDHSRDPGSLM-PVYSY-.....EPS-FLLPDDDVQGIQSLYG
GRP20 MMP13 GFLAHAFPPF.....EGIGGDAHFDDDETFSYRSP...... QYYNLFLVAAHEFGHSLGLEHSQDPGALMYPTYVYR.....DVDRFVLPRDDVNGIQSLYG
GRP21 MMP10 GVLAHAYAP-.....-GIGGDAHFDEDEQWTLGP...... -GAN-F-VAAHEFGHSLG-SHS-D-NALM-PT-SFG-TI..-PAQYKLSADDIAGIQTLYG
GRP22 MMP8 GILAHAFQPG.....QGIGGDAHFDAEETWT-TS-...... NYNLFLVAAHEFGHSLGLAHSSDPGALMYPNYAFR.....ETSNYSLPQDDI-GIQAIYG
GRP23 MMP8 GILAHAFQPG.....-GIGGDAHFDSEETWTQDSK...... NYNLFLVAAHEFGHSLGLSHSTDPGALMYPNYAYR.....EPSTYSLPQDDING-QTIYG
GRP24 MMP1 G-LAHA-QPG.....--IGGD-HFD----WT--F-...... -YNL-RVAAHE-GHSLGL-HSTDIGALMYP-Y---...... GDVQL-Q-DI-GIQAIYG
GRP25 MMP27 GVLGHAFPPG.....-GLGGDTHFDEDE-WTK.D-A...... GF-LFLVAAHEFGHALGLSHSNDQTALMFPNYVSL.....DP-KYPLSQDDI-GIQSIYG
GRP26 MMP27 GVLGHAFPPG.....LGLGGDTHFDEDETW---DGE...... GFNLFLVAAHEFGHSLGLSHS-DQTALMFPNY-SL.....DP-KYPLSQDDI-GIQSIYG
GRP27 MMP12 G--AHAFGPG.....-GIGGDAHFD-DEFWTTHS...... -GTNLFL-AVHE-GHSLGLGHSSDPKA-MFPTYKYV.....DINTF-LSADDIRGIQSLYG
GRP28 MMP12 GVIAHAFGPG.....PGIGGDTHFDEDEIWSKSY...... KGTNLFLVAVHELGHALGLDHSNDPKAIMFPTYGYI.....DLNTFHLSADDIRGIQSLYG
GRP29 MMP12 GTLAH-FYPG.....PGIQGD-HFDEAETWTKSF...... QGTNLFLVAVHELGHSLGL-HSNNPKSIM-PTYRY-.....-P-TFRLSADDI--IQSLY-
GRP30 MMP20 GTLAHAFAPG.....EGLGGDTHFDN-EKWTMG-N...... GFNLFTVAAHEFGHALGL-HSTDPSALM-PTYKY-.....-PY-F-LPKDDVKGIQALYG
GRP31 MMP7 NTLAHAFAPG.....-GLGGDAHFDEDERWTDGSS...... LGINFL-AATHELGHSLGMGHSSDP------.....------DDIKGIQKLYG
GRP32 MMP7 NTLGHAFAPG.....PGLGGDAHFDKDEYWTD-ED...... -GVNFLF-ATHE-GHSLGL-HSSVP--VMYPTYQ-D.....-SEDFSLTKDDIAGIQKLYG
GRP33 GVLAHANSPG.....PEQGGDTHFDDDEKWSLSSH...... NINLLLVAAHEFGHALGLDHSRDSSALMYPTYRYV.....STN-YTLPRDDRLGVQALYG
GRP34 MT1MMP GFLA-AYFPG.....PN--G------NED...... LNGND-FLVAVHELGHALGLEHS-DPS-IM-PFYQWM.....DTE-F-LP-D-RRGIQQLYG
GRP35 MT1MMP GFLAHAYFPG.....-GIGGDTHFDSAEPWTARN-D...... L-GNDLFLVAVHELGHALGLEHSNDPSAIMAPFYQWM.....DT-NFQLPDDDRRGIQQLYG
GRP36 MTMMP GFLAHAYFPG.....-GIGGDTHFDAAEPWT--N-D...... L-GND-FLVAVHELGHALGLEHSND-SAIMAPFYQWM.....-TENF-LPDDDRRGIQQ-YG
GRP37 MTMMP GFLAHAYFPG.....NGIGGDTHFDAAEPWTIGNKD...... LLGNDVFLVAVHELGHALGMEHSNDPSAIMAPFYQWM.....ETDHFVLPDDDRKGIQKLYG
GRP38 MTMMP,28 GFLAHAYFPG.....PGMGGDTHFDSDEPWTIG-QN...... -QGNDLFLVAVHELGHALGLEHSNNPLAIMAPFYQWM.....-T-NF-LP-DD--G-Q-IYG
GRP39 MT5MMP GFLAHAYFPG.....PGIGGDTHFDSDEPWTLGN-N...... HDGNDLFLVAVHELGHALGLEHS-DPSAIMAPFYQ-M.....ETHNFKLP-DDLQGIQKIYG
GRP40 MT3MMP GFLAHAYFPG.....PGIGGDTHFDSDEPWTLGNPN...... HDGNDLFLVA-HELGHALGLEHSNDP-AIMAPFYQYM.....-T-NF-LP-DDL-GIQKIYG
GRP41 MT2MMP GFLAHAYFPG.....PGLGGDTHFDADEPWTFSSTD...... LHG--LFLVAVHELGHALGLEHSSNP-AIMAPFYQW-.....D-D-F-LPEDDLRGIQQLYG
GRP42 MMP19 RVLAHADIPE...... LGSVHFDEDE-WTE-T...... Y-GVNLRIIAAHE-GHALGLGHSRY-QALMAPVY-GY-P...... -FKLHPDDVAGIQALYG
GRP43 KVLAHAD-PE...... LGS-HFD-DE-WTEGT...... YQGVNLRIIAAHEVGHALGLGHSRYTQALMAPVYAGYQP...... -F-LHPDDVAGIQALYG
GRP44 MMP26 GILGHAFLPN.....SGNPGVVHFDKNEHWS-S-...... TGYNLFLVATHEIGHSLGLQHSGN-SSIMYPTYWYH.....DP-TFQLSADDIQRIQHLYG
GRP45 MMP-28 GAL-HAFLPR...... RGEAHFD-DERWSLSR...... RRGRNLFV-LAHEIGHTLGL-HSPAPRALMAPYYK-LGR...... DALLSWDDVLAVQ-LYG
GRP46 MT6MMP GTLAHAFFPG...EHP.ISGDTHFDDEETWTFGS-DGE...... GTDLFAVAVHEFGHALGLGHSSAPNSIMRPFYQGP...VG-PDKYRLS-DDRDGLQQLYG
GRP47 MT6MMP GTLAHAFFPG...EHP.ISGDTHFDDEETWT-GSKDGE...... GTDLFAVAVHEFGHALGLGHSSAP-SIMRPFYQGP...VG-P-EYRLS-DDREGLQQLYG
GRP48 MT6MMP GTLAHAFFPG...EHP.ISGDTHFDDEETWTFGSTD--...... G-DLFAVAVHEFGHALGLGHSSAP-SIMRPFYQGP...VGDP-TYRL-QDDRDGLQQLYG
GRP49 MT4MMP GTVAHAFFPG...DHH.TAGD-HFDDDE-W-FRSSDAH...... -MDLFAVAVHEFGHAIGLSHVAA--SIMQPYYQGP...VGDP--Y-LPYEDRVR-WQLYG
GRP50 MT4MMP GTVAHAFFPG...---.TAGDTHFDDDE-WTFR--D-H...... GMDLFAVAVHEFGHAIGL-H-SAIESIMRPYYQGP...VGDPLKY-L-YEDKVR-WQLYG
GRP51 GTVAHAFFPG...EKF.TAGDTHFDDDEAWTFRSPEAD...... GMDLFAVAVHEFGHAIGLVHAA-VDSIMRPYYQGP...VGDPLKYDLPYEDKVRVWQLYG
GRP52 MMP2-likeQVLAHAFYPG.....EGRGGDAHFDADETWNFDGE...... SDDSHGTNFLNVALHELGHSLGLAHSAIPDAVMFPWYQNNE...... VAGNLPDDDRYGIQQLYG
GRP53 GVLAHATMPE...... SGMFHFDDDENWTYKDARKI..H..-N-ATDLLAVAIH-G-HTLGLEHSRDENAIMAPFYQKTTDS-GNYVYPNLKSDDISAIQAIYG
GRP54 N-LAHAFYPYEM-...-YGGD-HFDEDENWKENST...... HLS-GVDFYSVAIHELGHSLGLAHSPVYSSLMFPYYKGI-QG...... TLDYDDILA-Y-LY-
SUMMARY XxXXHaXXPX x dxhfdxXe wxxxx XXXXXXXaXHexgHXlGlxhXxxx XXmxpxyxxx x lx dx XxXXXy
Fig. S5. BINDSIght scoring of potential importance to specificity. Cumulative scores of evidences of specificity for solubilized elastin or α1(V) THP substrates are displayed in panels (a) and (b), respectively. A sequence position received a score of 1 if it distinguishes subfamilies of MMPs (is class-specific) or 0.5 if nearly class-specific. Conserved positions were penalized to remove them from consideration. In panel (a), a score of 2 was added if a residue is among the top 10%-ile most protected by Eln-20 (see Figure 2a), a score of 1 if among the next 15%-ile most protected, or a score of 0.5 if among the third tier of protection (20%-ile wide). In panel (a), a score of 1 was added if Eln-20 shifts the NMR peak such that ΔωHN ≥ 0.04 ppm or if it broadens the peak in the top 10%-ile (see Figure 1a). A score of 0.5 was added if 0.04 ppm > ΔωHN > 0.02 ppm. In panel (b), a score of 2 was added if a residue is protected by the α1(V) 436-447 THP from Gd(III)·EDTA by ≥ 0.4 (see Figure 4c of ref (1)) or a score of 1 if protected ≥ 0.15 but < 0.4. In panel (b), an NMR peak shift by α1(V) 436-447 THP (see Figure 2 of ref (1)) added 1 to the score if ΔωHN ≥ 0.029 ppm or 0.5 if 0.029 ppm > ΔωHN ≥ 0.022 ppm. Surface residues around the periphery of the active site selected for mutagenesis are listed. “b” denotes a buried side chain.
REFERENCE
1.Bhaskaran, R., Palmier, M. O., Lauer-Fields, J. L., Fields, G. B., and Van Doren, S. R. (2008) J. Biol. Chem.283, 21779-21788
Fig.S6.Location of residues identified by BINDSIght in the catalytic domain of MMP-12.Panels a and b refer to Fig. S5 panels a and breporting BINDSight analyses with elastin fragments and the α1(V) 436-447 THP, respectively. Cyan marksresidues that are both protected by the substrate and class-specific in ET analysis. Gray-bluemarks those with chemical shift perturbed by the substrate and class-specific in ET analysis. Blue indicates residues having all three properties. Purple residues such as conserved M236 are both protected and perturbed in chemical shift by the substrate.
apparent
kcat/Km,
M-1s-1 / 34,580
± 690 / 14,040
± 30 / 33,030
± 40 / 33,710
± 40 / 29,950
±110 / 30,540
± 40 / 13,440
± 50 / 12,440
± 140 / 6090
± 20 / 28,010
± 170 / 41,130
± 290 / 24,040
± 70 / 9780
± 160 / 10,650
± 30
(kcat/Km)mut
(kcat/Km)WT / 41% / 95.5% / 97.5% / 87% / 88% / 39% / 36% / 18% / 81% / 119% / 69.5% / 28% / 31%
TableS1. Apparent catalytic efficiencies of MMP-12 variants toward DQ-collagen I1 at 25 ºC, pH 7.5.
1 The susceptibility of DQ-collagen I to trypsin or MMP-3 cat dom suggests it to be less completely triple helical thanthe triple helix of unlabeled collagen I. The heterogeneity in molecular weight of DQ-collagen I species is likely to affect the population of sites accessible to proteolysis. Nonetheless, the fitting of this quantitative comparison of catalytic efficiency as apparentkcat / Kmrelies instead on the accuracy of the enzyme concentration, which has been determined carefully by active site titration.
2 Wild-type represents an average of numerous repetitions.
Table S2. Steady-state enzyme kinetics parameters of combined mutations
in V-B loop of MMP-12 at 25 ºC, pH 7.5.
Substrate / Wild-type / T205K, H206D / T205K, H206D,S207T, G208T
general substrate FS-6 / kcat/Km,M-1s-1 / 133,800
± 6000 / 118,300
± 300 / 120,000
± 300
ΔΔG‡,1kcal/mol / 0.00 / 0.007 / 0.06
kcat, s-1 / 17.4 ± 0.6 / 17.6 ± 1.0 / 17.4 ± 2.6
Km,μM / 130 ± 4 / 149 ± 5 / 150 ± 22
fELN-125 / kcat/Km,M-1s-1 / 10,200
± 400 / 5730
± 20 / 5180
± 10
ΔΔG‡,1kcal/mol / 0.00 / 0.34 / 0.40
kcat, s-1 / 0.0105 ± 0.0003 / 0.0081 ± 0.0002 / 0.0114 ± 0.0004
Km,μM / 1.1 ± 0.02 / 1.4 ± 0.04 / 2.2 ± 0.08
α1(V) 436-447 fTHP / kcat/Km,M-1s-1 / 55,400 ± 350 / 16,700 ± 200 / 17,700 ± 400
ΔΔG‡,1kcal/mol / 0.00 / 0.71 / 0.68
kcat, s-1 / 1.2 ± 0.10 / 1.2 ± 0.20 / 1.1± 0.20
Km,μM / 22 ± 5.6 / 70 ± 12.0 / 60 ± 7.0
DQ-collagen I / kcat/Km,M-1s-1 / 34,580
± 690 / 27,230
± 110 / 26,380
± 80
ΔΔG‡,1kcal/mol / 0.00 / 0.14 / 0.16
DQ-collagen IV / kcat/Km,M-1s-1 / 29,200
± 190 / 26,160
± 100 / 26,700
± 80
ΔΔG‡,1kcal/mol / 0.00 / 0.07 / 0.05
1 ΔΔG‡ = -RT·ln[(kcat/Km)mutant / (kcat/Km)wildtype] at 298 K