Membrane Type-1 Matrix Metalloprotease and Stromelysin-3 Cleave More Efficiently Synthetic Substrates Containing Unusual Amino Acids in Their P1′ Positions
1998; Elsevier BV; Volume: 273; Issue: 5 Linguagem: Inglês
10.1074/jbc.273.5.2763
ISSN1083-351X
AutoresArtur Mucha, Philippe Cuniasse, Rama Kannan, Fabrice Beau, Athanasios Yiotakis, Paul Basset, Vincent Dive,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe influence of the substrate P1′ position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH2, where Xaa in the P1′ position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P1′ position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the kcat/Km ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with aS-para-methoxybenzyl cysteine residue in the P1′ position displayed akcat/Km value of 1.59 106m−1 s−1 and 1.67 104m−1 s−1, when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generatedin vivo by some post-translational modifications. The influence of the substrate P1′ position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH2, where Xaa in the P1′ position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P1′ position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the kcat/Km ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with aS-para-methoxybenzyl cysteine residue in the P1′ position displayed akcat/Km value of 1.59 106m−1 s−1 and 1.67 104m−1 s−1, when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generatedin vivo by some post-translational modifications. Matrix metalloproteases (MMPs), 1The abbreviations used are: MMP, matrix metalloprotease; MT1, membrane type-1; ST, stromelysin; COL, collagenase; dns, dansyl, 5-dimethylaminonaphthalene-1-sulfonyldansyl; Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N3-(2,4-dinitrophenyl)-l-2,3-diamino propionyl; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate; Bzl, benzyl; Cys(OMeBzl), S-para-methoxybenzyl cysteine; HPLC, high performance liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl. 1The abbreviations used are: MMP, matrix metalloprotease; MT1, membrane type-1; ST, stromelysin; COL, collagenase; dns, dansyl, 5-dimethylaminonaphthalene-1-sulfonyldansyl; Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N3-(2,4-dinitrophenyl)-l-2,3-diamino propionyl; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate; Bzl, benzyl; Cys(OMeBzl), S-para-methoxybenzyl cysteine; HPLC, high performance liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl. also known as matrixins, form a group of structurally related zinc endopeptidases collectively able to degrade all components of the extracellular matrix (1Coussens L.M. Werb Z. Chem. Biol. 1996; 3: 895-904Abstract Full Text PDF PubMed Scopus (499) Google Scholar). MMPs are believed to be mediators of both normal and pathological tissue remodeling processes, and their increased expression has been observed in a variety of human disorders (2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2619) Google Scholar, 3MacDougall J.R. Matrisian L.M. Cancer Metastasis Rev. 1995; 14: 351-362Crossref PubMed Scopus (399) Google Scholar, 4Stetler-Stevenson W.G. Hewitt R. Corcoran M. Cancer Biol. 1996; 7: 147-154Crossref PubMed Scopus (339) Google Scholar). In particular, membrane type-1 (MT1)-MMP, a progelatinase A activator (5Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2361) Google Scholar), and stromelysin-3 (ST3), a matrixin with unusual functional properties (6Basset P. Bellocq J.P. Lefebvre O. Noël A. Chenard M.P. Wolf C. Anglard P. Rio M.C. Crit. Rev. Oncol-Hematol. 1997; 26: 43-53Crossref PubMed Scopus (69) Google Scholar), are expressed in most human carcinomas (7Rouyer N. Wolf C. Chenard M.P. Rio M.C. Chambon P. Bellocq J.P. Basset P. Invasion Metastasis. 1994; 95: 269-275Google Scholar, 8Okada A. Bellocq J.P. Rouyer N. Chenard M.P. Rio M.C. Chambon P. Basset P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2730-2734Crossref PubMed Scopus (486) Google Scholar, 9Sato H. Seiki M. J. Biochem. (Tokyo). 1996; 119: 209-215Crossref PubMed Scopus (195) Google Scholar). MT1-MMP and ST3 both belong to a subgroup of MMPs which are believed to be intracellularly activated by furin or furin-like convertases, thereby suggesting that MT1-MMP and ST3 are present in tissues in an active form, in contrast to other MMPs which are secreted as inactive zymogens (10Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (527) Google Scholar, 11Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 12Nagase H. Biol. Chem. Hoppe-Seyler. 1997; 378: 151-160PubMed Google Scholar). As part of a program aimed at developing therapeutic inhibitors for these two MMPs, the specificity of these enzymes in cleaving synthetic substrates has been investigated. Current data on the specificity of MT1-MMP toward the degradation of synthetic substrates are extremely limited (13Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar), while no synthetic substrate has yet been reported to be cleaved by ST3. In contrast to MT1-MMP and ST3, several studies have been devoted to the delineation of the specificity of other matrixin family members by developing both synthetic substrates (14Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (669) Google Scholar, 15Niedzwiecki L. Teahan J. Harrison R.K. Stein R.L. Biochemistry. 1992; 31: 12618-12623Crossref PubMed Scopus (73) Google Scholar, 16Netzel-Arnett S. Sang Q.X. Moore W.G.I. Navre M. Birkedal-Hansen H. Van Wart H.E. Biochemistry. 1993; 32: 6427-6432Crossref PubMed Scopus (161) Google Scholar, 17Nagase H. Fields C.G. Fields G.B. J. Biol. Chem. 1994; 269: 20952-20957Abstract Full Text PDF PubMed Google Scholar, 18Welch A.R. Holman C.M. Browner M.F. Gehring M. Kan C.C. Van Wart H.E. Arch. Biochem. Biophys. 1995; 324: 59-64Crossref PubMed Scopus (28) Google Scholar) and inhibitors for these enzymes (19Hagmann W.K. Lark M.W. Becker J.W. Annu. Rev. Med. Chem. 1996; 31: 231-240Google Scholar, 20Beckett R.P. Exp. Opin. Ther. Patents. 1996; 6: 1305-1315Crossref Google Scholar). Such approaches have been greatly facilitated by the resolution of the crystal structures of several MMP catalytic domains (21Lovejoy B. Cleasby A. Hassel A.M. Longley K. Luther M.A. Weigl D. McGeehan G. McElroy A.B. Drewry D. Lambert M. Jordan S.R. Science. 1994; 263: 375-377Crossref PubMed Scopus (304) Google Scholar, 22Bode W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (322) Google Scholar, 23Lovejoy B. Hassel A.M. Luther M.A. Weigl D. Jordan S.R. Biochemistry. 1994; 33: 8207-8217Crossref PubMed Scopus (153) Google Scholar, 24Borkakoti N. Winkler F.K. Williams D.H. D'Arcy A. Broadhurst M.J. Brown P.A. Johnson W.H. Murray E.J. Nat. Struct. Biol. 1994; 1: 106-110Crossref PubMed Scopus (210) Google Scholar, 25Stams T. Spurlino J.C. Smith D.L. Wahl R.C. Ho T.F. Qoronfleh M.W. Banks T. Rubin B. Nat. Struct. Biol. 1994; 1: 119-123Crossref PubMed Scopus (200) Google Scholar, 26Spurlino J.C. Smallwood A.M. Carlton D.D. Banks T.M. Vavra K.J. Johnson J.S. Cook E.R. Falvo J. Wahl R.C. Pulvino T.A. Wendoloski J.J. Smith D.L. Proteins Struct. Funct. Genet. 1994; 19: 98-109Crossref PubMed Scopus (194) Google Scholar, 27Browner M.F. Smith W.W. Castelhano A.L. Biochemistry. 1995; 34: 6602-6610Crossref PubMed Scopus (248) Google Scholar), allowing structure-based design strategies to be used (28Esser C.K. Bugianesi R.L. Caldwell C.G. Chapman K.T. Durette P.L. Girotra N.N. Kopta I.E. Lanza T.J. Levorse D.A. MacCross M. Owens K.A. Ponpipom M.M. Simeone J.P. Harrison R.K. Niedzwiecki L. Becker J. Marcy A.I. Axel M.A. Christen A.J. McDonnell J. Moore V.L. Olszewski J.M. Saphos C. Visco D.M. Shen F. Colleti A. Krieter P.A. Hagmann W.K. J. Med. Chem. 1997; 40: 1026-1040Crossref PubMed Scopus (63) Google Scholar). According to these structural studies, the S1′ subsite of these enzymes appears as a cavity of variable size, depending on the nature of the amino acid residue located near the bottom of this cavity. In stromelysin-1 (ST1), which contains leucine in this particular position, the S1′ pocket is a deep cavity, forming a channel that extends through the whole body of the enzyme catalytic domain (29Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M.D. Cameron P.M. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (269) Google Scholar, 30Dhanaraj V. Ye Q-Z Johnson L.L. Hupe D.J. Ortwine D.F. Dunbar J.B. Rubin J.R. Pavlocski A. Humblet C. Blundell T.L. Structure. 1996; 4: 375-386Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). X-ray structure analysis of a complex between ST1 and a carboxylalkyl inhibitor harboring a homophenylalanine in the P1′ position has consistently shown that the homophenylalanine side chain only fills half of the S1′ pocket of ST1 (29Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M.D. Cameron P.M. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (269) Google Scholar). A comparable situation is likely to occur in most other MMPs, including MT1-MMP, collagenase-2 (COL2), stromelysin-1 (ST1), stromelysin-2 (ST2), gelatinase A, and gelatinase B, due to the presence in their S1′ pocket of a leucine at the same position. The high potency of several inhibitors, substituted in their P1′ position by side chains longer than homophenylalanine, toward this subgroup of matrixins is consistent with this proposal (28Esser C.K. Bugianesi R.L. Caldwell C.G. Chapman K.T. Durette P.L. Girotra N.N. Kopta I.E. Lanza T.J. Levorse D.A. MacCross M. Owens K.A. Ponpipom M.M. Simeone J.P. Harrison R.K. Niedzwiecki L. Becker J. Marcy A.I. Axel M.A. Christen A.J. McDonnell J. Moore V.L. Olszewski J.M. Saphos C. Visco D.M. Shen F. Colleti A. Krieter P.A. Hagmann W.K. J. Med. Chem. 1997; 40: 1026-1040Crossref PubMed Scopus (63) Google Scholar,31Morphy J.R. Beeley N.R.A. Boyce B.A. Leonard J. Mason B. Millican A. Millar K. O'Connell J.P. Porter J. Bioorg. Med. Chem. Lett. 1994; 4: 2747-2752Crossref Scopus (40) Google Scholar, 32Wahl R.C. Pulvino T.A. Mathiowetz A.M. Ghose A.K. Johnson J.S. Delecki D.D. Cook E.R. Gainor J.A. Gowravaram M.R. Tomczuk B.E. Bioorg. Med. Chem. Lett. 1995; 5: 349-352Crossref Scopus (17) Google Scholar, 33Sahoo P.P. Caldwell C.G. Chapman K.T. Durette P.L. Esser C.K. Kopka I.E. Polo S.A. Sperow K.M. Niedzwiecki L.M. Izquierdo-Martin M. Chang B.C. Harrison R.K. Stein R.L. MacCoss M. Hagmann W.R. Bioorg. Med. Chem. Lett. 1995; 5: 2441-2446Crossref Scopus (27) Google Scholar, 34Tomczuk B.E. Gowravaram M.R. Johnson J.S. Delecki D.D. Cook E.R. Ghose A.K. Mathiowetz A.M. Spurlino J.C. Rubin B. Smith D.L. Pulvino T.A. Wahl R.C. Bioorg. Med. Chem. Lett. 1995; 5: 343-348Crossref Scopus (18) Google Scholar, 35Tamaki K. Tanzawa K. Kurihara S. Oikawa T. Monma S. Shimada K. Sugimura Y. Chem. Pharm. Bull. 1995; 43: 1883-1893Crossref PubMed Scopus (38) Google Scholar). In contrast to this subgroup of matrixins, collagenase-1 (COL1), matrilysin, and ST3 possess in their S1′ subsite a residue other than leucine. COL1, matrilysin, and ST3 possess in this particular position an arginine, a tyrosine and a glutamine, respectively. In the case of this matrixin subgroup, x-ray structures of COL1 and matrilysin have demonstrated that the presence of either arginine or tyrosine reduces the size of their S1′ subsite (24Borkakoti N. Winkler F.K. Williams D.H. D'Arcy A. Broadhurst M.J. Brown P.A. Johnson W.H. Murray E.J. Nat. Struct. Biol. 1994; 1: 106-110Crossref PubMed Scopus (210) Google Scholar, 26Spurlino J.C. Smallwood A.M. Carlton D.D. Banks T.M. Vavra K.J. Johnson J.S. Cook E.R. Falvo J. Wahl R.C. Pulvino T.A. Wendoloski J.J. Smith D.L. Proteins Struct. Funct. Genet. 1994; 19: 98-109Crossref PubMed Scopus (194) Google Scholar, 27Browner M.F. Smith W.W. Castelhano A.L. Biochemistry. 1995; 34: 6602-6610Crossref PubMed Scopus (248) Google Scholar). While no crystal structure is presently available for ST3, a similar situation has been suggested to occur in this matrixin (36Okada A. Saez S. Misumi Y. Basset P. Gene (Amst.). 1997; 185: 187-193Crossref PubMed Scopus (20) Google Scholar). While the particular shape of the S1′ pocket in matrixins has been extensively exploited for the design of matrixin inhibitors, only one study so far examined the influence of this S1′ cavity on the cleavage of synthetic substrates with unusual amino acids in their P1′ position (37McGeehan G.M. Bickett D.M. Green M. Kassel D. Wiseman J.S. Berman J. J. Biol. Chem. 1994; 269: 32814-32820Abstract Full Text PDF PubMed Google Scholar). It is worth remembering that most of the matrixin inhibitors developed to date and harboring long side chains in their P1′ position are not transition-state analogues. Therefore, the ability of matrixins to cleave substrates containing in their P1′ position amino acids with unusual side chains cannot be predicted from these inhibitor studies. To address this issue more systematically in the case of MT1-MMP and ST3, synthetic heptapeptides containing in their P1′ position amino acids with arylalkyl or alkyl side chains of varying size were synthesized. The amino acid sequence covering the P3 to P3′ positions of these substrates was selected according to the canonical -Pro-Leu-Gly(Ala)-Leu-Trp-Ala- sequence previously established for matrixins (38Stack M.S. Gray R.D. J. Biol. Chem. 1989; 264: 4277-4281Abstract Full Text PDF PubMed Google Scholar, 39Netzel-Arnett S. Mallya S.K. Nagase H. Birkedal-Hansen H. Van Wart H.E. Anal. Biochem. 1991; 195: 86-92Crossref PubMed Scopus (106) Google Scholar). In these substrates, the cleavage site has been demonstrated to occur between the Gly(Ala)-Leu residues. Accordingly, several fluorogenic substrates were prepared by substituting the N-terminal side of this canonic sequence with a dansyl group (dns), as a quencher, the tryptophan in the P2′ position of this sequence being retained as a fluorophore. An arginine residue was added at the C-terminal extremity of this sequence to improve peptide solubility. In the present report, these fluorogenic peptides were evaluated as substrates for MT1-MMP and ST3. Based on this study, a fluorogenic compound, containing dinitrophenyl-coumarin as a quencher-fluorophore pair, was also synthesized and examined for comparison. Rink amide resin, 2-(1H-benzotriazol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate (HBTU) and all Fmoc natural amino acid derivatives were purchased from Novabiochem. The unusual Fmoc amino acids were from Novabiochem and Advanced Chemtech. 7-Methoxycoumarin-2-acetic acid (McaOH) and 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride, dnsCl) were from Aldrich. Unusual Fmoc amino acids not commercially available (namely, 2-aminoheptanoic acid and 2-amino-5-phenyl-pentanoic acid) were synthesized by catalytic phase transfer alkylation of ethyl diphenylmethyleneglycinate with the appropriate alkyl bromides followed by hydrolysis, as described by O'Donnell et al. (40O'Donnell M.J. Boniece J.M. Earp S.E. Tetrahedron Lett. 1978; 30: 26-41-2644Google Scholar) and O'Donnell and Eckrich (41O'Donnell M.J. Eckrich T.M. Tetrahedron Lett. 1978; 47: 4625-4628Crossref Scopus (110) Google Scholar). 2-Aminoheptanoic, 2-aminooctanoic (Aldrich) and 2-amino-5-phenyl-pentanoic acids were converted into their Fmoc derivatives by the action of Fmoc-Cl in water/Na2CO3/dioxane solution according to the standard literature procedure (42Carpino L.A. Han G.H. J. Org. Chem. 1972; 37: 3404-3409Crossref Scopus (1080) Google Scholar). The Fmoc amino acids were obtained as white, crystalline compounds after their purification by flash chromatography on silica gel using ethyl acetate/hexane/acetic acid eluents. Their structure and the purity were confirmed by NMR and mass spectroscopy analysis. These unusual Fmoc amino acids were used for peptide synthesis as mixtures of enantiomers. For these peptides, the diastereoisomers were separated by HPLC. N2-Fmoc-N3-2,4-dinitrophenyl-l-2,3-diaminopropionic acid (Fmoc-DpaOH) was synthesized starting from Fmoc-l-AsnOH, as described by Knight et al.(14Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (669) Google Scholar). Solid phase synthesis of the substrates was performed in a model 357 Advanced Chemtech multiple peptide synthesizer on a Rink amide resin. Typically, three equivalents of an Fmoc amino acid, three equivalents of HBTU, and five equivalents of diisopropylethylamine inN-methylpyrrolidone were added to the resin, and the coupling reaction was allowed to proceed for 30 min. The FmocN-protection group was removed with a 30% solution of piperidine in N-methylpyrrolidone. N-terminal acylation of the peptides was achieved either with excess dansyl chloride (20 equivalents) in the presence of diisopropylethylamine or with triple coupling of McaOH, under the conditions described above. Cleavage of the peptides from the resin, together with the cleavage of the side chain protection groups, were performed by the action of trifluoroacetic acid containing 5% triisopropylsilane. All peptides were purified by preparative HPLC column (Vydac, 218TP1022) performed on a Gilson system equipped with a variable wavelength detector. Gradient elutions were performed using solutions A (10% acetonitrile in 0.1% trifluoroacetic acid in water) and B (90% acetonitrile in 0.1% trifluoroacetic acid in water). All peptides were recovered by lyophilization. Peptide purities were checked by amino acid analyses, analytical HPLC (Vydac, 218TP104 column) and mass spectroscopy. cDNAs corresponding to the catalytic domains of mouse ST3 (Phe-102 to Ser-276) and human MT1-MMP (Tyr-111 to Arg-298) were introduced into the expression vector pET-3b, expressed inEscherichia coli BL21 (DF3) cells after isopropyl-1-thio-β-d-galactopyranoside induction and purified essentially as described in Noël et al. (43Noël A. Santavicca M. Stoll I. L'Hoir C. Staub A. Murphy G. Rio M.C. Basset P. J. Biol. Chem. 1995; 270: 22866-22872Crossref PubMed Scopus (64) Google Scholar). Briefly, both MMP catalytic domains were solubilized from bacterial inclusion bodies with 8 m urea in the presence of 100 mm dithiothreitol and purified on a Q-Sepharose anion-exchange column (Pharmacia Biotech Inc.). Purified catalytic domains were then slowly refolded at a protein concentration of 50 μg/ml by dialysis to dilute out the urea. The refolding step was followed by size exclusion chromatography, using a gel filtration column (Superdex-gf 200, Pharmacia) to eliminate the aggregates, and to retain the active monomeric protein alone. Substrate specificity assays were performed in 50 mm Tris/HCl buffer, pH 7.5, 10 mmCaCl2, in the absence (MT1-MMP) or presence of 0.2m NaCl (ST3), at 25 °C. Substrate concentrations were determined spectrophotometrically using ε340 nm = 4300m−1 cm−1 for dansyl peptides (44Stöcker W. Ng M. Auld D.S. Biochemistry. 1990; 29: 10418-10425Crossref PubMed Scopus (59) Google Scholar) and ε328 nm = 12900 m−1cm−1 for coumarin peptides (14Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (669) Google Scholar). Substrates were prepared as 1 mm stock solutions in dimethyl sulfoxide. Enzyme concentrations were determined from optical density, using the method of Gill and von Hippel (45Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5009) Google Scholar) to calculate the extinction coefficient of these two matrixins. In the case of MT1-MMP, values of kcat/Km were determined from first-order full-time course reaction curves obtained at [S] ≪Km (S = 0.2 μm), at 10 nm final enzyme concentration. These progress curves were monitored by following the increase in fluorescence at 340 nm (λex = 280 nm), induced by the cleavage of the dns substrates, in a Biologic PMS 200 spectrophotometer. Due to the lower efficiency of ST3 in cleaving this series of substrates, the observation of full-time course reactions for ST3 has required the use of higher enzyme concentrations, leading to a high fluorescence background. Thus, the kinetic parameters for ST3 were based on HPLC (Thermo Separation Products system) allowing the separation of the unreacted substrate from the cleavage products and its quantification. Immediately after the initiation of the reaction, aliquots were withdrawn from this reaction solution by the autosampler (Spectra System AS300), at predetermined time intervals, and injected onto the column. Substrate and products (S = 0.2 μm, ST3 concentration from 50 to 200 nm) were separated on a C18 column (Vydac, 218TP104), eluted with a linear acetonitrile gradient in 0.1% trifluoroacetic acid. Products were detected using an FL300 Spectra System fluorescence detector (λex, 280 nm; λem, 340 nm) and were identified by mass spectroscopy analysis. Data analysis was performed with a Thermo Separation Products datajet integrator. The two approaches used for MT1-MMP and ST3 yielded product progress curves, from which values for the specificity constantkcat/Km were determined by fitting these curves with the integrated Michaelis-Menten Equation 1, by nonlinear regression (46Wahl R.C. Anal. Biochem. 1994; 219: 383-384Crossref PubMed Scopus (12) Google Scholar) [P]=[S0](1−exp(−kt))Equation 1 where k = (kcat/Km)·Et = [total enzyme]. For eachkcat/Km determination, three independent experiments were taken into consideration. Individual kinetic parameters (kcat and Km) for MT1-MMP and ST3 were obtained from analysis of fluorescence curves, under steady-state rate conditions, over 0.2 to 5 Km substrate concentration ranges. The experiments were carried out in a 5 × 5 × 45-mm fluorescence cell, using excitation at 280 or 300 nm (slit width, 5 nm) and emission at 360 nm (slit width, 5 nm) for the dns derivatives, and excitation at 328 nm (slit width, 5 nm) and emission at 400 nm (slit width, 5 nm) for the Mca derivatives. Cleavage of the following substrates: dns-Pro-Leu-Ala-Leu-Trp-Ala-Arg-NH2, dns-Pro-Leu-Ala-Cys(OMeBzl)-Trp-Ala-Arg-NH2, and Mca-Pro-Leu-Ala-Cys(OMeBzl)-Trp-Ala-Arg-Dpa-NH2, was accompanied by a 12-, 13-, and 16-fold increase in fluorescence, respectively. Initial rates measurements for twelve different substrate concentrations were performed. Km and kcat values were determined by fitting these data to the equation of Michaelis-Menten by nonlinear regression analysis. Depending upon the concentration of substrate used in a typical assay, it is possible that fluorescence measurements may be affected by an inner filter effect. To determine the extent of this effect in the case of the dns-derivatives, the fluorescence intensity of a 10 μm Leu-Trp-Ala-Arg-NH2 solution was measured in the presence of several dns-Pro-Leu-Ala-Leu-Trp-Ala-Arg-NH2 substrate concentrations (from 1 μm to 50 μm). From these experiments, a correction factor for each substrate concentration was deduced and used to correct the fluorescence observed at high substrate concentrations for the inner filter effect. The same approach was used for the Mca derivatives, using a 1 μmMca-Pro-Leu-Ala solution as the fluorescence standard. The different side chains in the P1′ position of our series of fluorogenic substrates are described in Fig. 1. Arylalkyl side chains longer than that of phenylalanine were selected to assess the effect of the side chain's length on MT1-MMP1 and ST3 cleaving activities. In addition, substrates with arylalkyl side chains containing an oxygen or sulfur heteroatom were also examined. The effects of alkyl side chains longer than leucine were also studied. Products resulting from the cleavage of this series of substrates by MT1-MMP or ST3 were characterized both by HPLC and mass spectroscopy. For each substrate, we observed a single cleavage site between the expected Ala-Xaa peptide bond, with no inhibition of the reaction by the released products (Fig. 2, and data not shown). Values of the specificity constantkcat/Km that were determined from the first-order progress curves for the hydrolysis of substrates by MT1-MMP are reported in Table I. The most rapidly cleaved substrate by this enzyme was that harboring the longest side chain in the P1′ position (Cys(OMeBzl)). In the arylalkyl series, the lengthening of the side chain by one (hPhe) and two (pPhe) methylene(s) resulted in a marked increase in catalytic efficiency, as compared with the substrate containing phenylalanine in the P1′ position. The comparison of substrates with a side chain of equal length (pPhe, Ser(Bzl), Cys(Bzl)) reveals the significant role played by a sulfur atom in the γ position of the side chain, which was associated with a much faster cleavage rate of the substrate by MT1-MMP. In the alkyl series, the rate of hydrolysis was also found to depend on the length of the side chain in the P1′ position. Comparison between the arylalkyl and alkyl series of substrates reveals that, in addition to the side chain length, the presence of the phenyl aromatic group is a determinant factor for the optimization of the rate of hydrolysis, since the substrates in the alkyl series were always cleaved at a slower rate than their counterparts in the arylalkyl series (Table I). The substitution Leu → Cys(OMeBzl) in these substrates led to a 37-fold increase in the kcat/Km ratio (Table I). In the case of ST3, the preferred side chain in the P1′ position of the substrate was also the longest arylalkyl one (Cys(OMeBzl)) (Table I). However, the substrates containing leucine, methionine or n-pentyl in the P1′ position were cleaved more rapidly than that with ann-hexyl side chain. As compared with MT1-MMP, the former three substrates were rather well cleaved by ST3, while the n-hexyl compound was a poor substrate.Table IComparison of the MT1-MMP and ST3 specificitySubstrateskcat/KmMT1-MMPST3m−1 s−1 (104)Dns-Pro-Leu-Ala-Cys(OMeBzl)-Trp-Ala-Arg-NH2159 ± 0.441.67 ± 0.04Dns-Pro-Leu-Ala-Cys(Bzl)-Trp-Ala-Arg-NH265 ± 0.321.12 ± 0.09Dns-Pro-Leu-Ala-Ser(Bzl)-Trp-Ala-Arg-NH225 ± 0.050.70 ± 0.01Dns-Pro-Leu-Ala-pPhe-Trp-Ala-Arg-NH224 ± 0.070.18 ± 0.009Dns-Pro-Leu-Ala-hPhe-Trp-Ala-Arg-NH29.5 ± 0.060.27 ± 0.03Dns-Pro-Leu-Ala-nHex-Trp-Ala-Arg-NH26.3 ± 0.040.001 ± 0.004Dns-Pro-Leu-Ala-Leu-Trp-Ala-Arg-NH24.2 ± 0.030.18 ± 0.0015Dns-Pro-Leu-Ala-Met-Trp-Ala-Arg-NH23.6 ± 0.030.52 ± 0.006Dns-Pro-Leu-Ala-nPent-Trp-Ala-Arg-NH23.1 ± 0.020.30 ± 0.005Dns-Pro-Leu-Ala-Nle-Trp-Ala-Arg-NH21.8 ± 0.010.13 ± 0.005Dns–Pro-Leu-Ala-Phe-Trp-Ala-Arg-NH21.1 ± 0.010.02 ± 0.006Dns–Pro-Leu-Phe-Cys(Bzl)-Trp-Ala-Arg-NH2<10.52 ± 0.03Dns–Ala-Ala-Ala-Cys(Bzl)-Trp-Ala-Arg-NH2<10.90 ± 0.02 Open table in a new tab The Km, kcat and kcat/Km values determined for two substrates (Xaa = Leu and Cys(OMeBzl)) are reported in Table II. For these matrixins, the substitution Leu → Cys(OMeBzl) was found to increase both the substrate affinity and the kcat value. However, while these two substrates displayed similar Km for MT1-MMP and ST3, the kcat values determined for ST3 on these substrates were about two orders of magnitude lower than those measured for MT1-MMP. The free energy difference (ΔΔG‡) associated with the substitution Leu → Cys(OMeBzl) was evaluated for each enzyme from the ratio of the kcat/Km values determined for these two substrates. For MT1-MMP, this substitution corresponds to a free energy change of 2 kcal/mol, while the same modification causes a free energy change of 1.55 kcal/mol for ST3.Table IIKinetic parameters for the hydrolysis by MT1-MMP and ST3 of substrates containing a Cys(OMeBzl) or Leu side chain in their P1′ positionSubstrateMT1-MMPST3Kmkcatkcat/Km2-aCalculated from the ratio of the kcat and Km values.Kmkcatkcat/Km2-aCalculated from the ratio of the kcat and Km values.μms−1m−1 s−1μms−1m−1 s−1Xaa = Cys(OMeBzl)2.79 ± 0.84.3 ± 0.715412181.5 ± 0.170.033 ± 0.00322000Xaa = Leu13.7 ± 0.320.73 ± 0.01532846.8 ± 1.10.011 ± 0.0011617ΔΔG‡2-bΔΔG‡ = RTln[(kcat/Km)Cys(OMeBzl)/(kcat/Km)Leu], T = 298K. Leu → Cys(OMeBzl)2.00 kcal/mol1.55 kcal/mol2-a Calculated from the ratio of the kcat and Km values.2-b ΔΔG‡ = RTln[(kcat/Km)Cys(OMeBzl)/(kcat/Km)Leu], T = 298K. Open table
Referência(s)