Role of the S-1 ' subsite glutamine 215 in activity and specificity of stromelysin-3 by site-directed mutagenesis

The influence of Gln215 in stromelysin-3 (MMP-11), a residue located in the S-1' subsite, was determined by producing three single mutants of this pos ition. As compared to wild-type stromelysin-3, the kinetic parameters K-M a nd k(cat) for the degradation of the fluorogenic substrate Dns-Pro-Leu-Ala- Leu-Trp-Ala-Arg-NH2 (Dns-Leu) by these mutants indicated that the Gln/Leu s ubstitution led to a 4-fold decrease in catalytic efficiency, whereas the m utations Gln/Tyr and Gln/Arg increased this parameter by a factor 10, The c leavage of alpha 1-protease inhibitor (alpha 1-PI), a natural substrate of stromelysin-3, by these mutants was also determined. Their relative activit ies for the degradation of alpha 1-PI correspond to those observed with the synthetic substrate Dns-Leu. The catalytic efficiency of wild-type stromel ysin-3 and its mutants to cleave the P-1' analogue of Dns-Leu, containing t he unusual amino acid Cys(OMeBn) (Dns-Cys(OMeBn)), was also determined. The values of the specificity factor, calculated as the ratio (k(cat)/K-M)(Dns -Cys(OMeBn))/(k(cat)/K-M)(Dns-Leu), were observed to vary from 26 for the w ild-type stromelysin-3 to 120 for the Gln/Leu mutant and 25 for the Gln/Arg mutant. The Gln/Tyr mutant did not cleave the substrate when its PI positi on is substituted by the unusual amino acid Cys(OMeBn). Altogether these ob servations established that both the catalytic activity and the specificity of stromelysin-3 are dependent on the nature of the residue in position 21 5. Finally, the cleavage efficiency of the Dns substrates by three represen tative matrixins, namely, MMP-14 (215 Leu), MMP-1 (215 = Arg), and MMP-7 (2 15 Tyr), was determined. Interestingly, the trends observed for these enzym es were similar to those established for the three mutants of stromelysin-3 , pointing out the influence of position 215 toward the selectivity in this family of enzymes.