Expression, characterization and structure determination of an active sitemutant (Glu202-Gln) of mini-stromelysin-1

Human stromelysin-1 is a member of the matrix metalloproteinase (MMP) famil y of enzymes. The active site glutamic acid of the MMPs is conserved throug hout the family and plays a pivotal role in the catalytic mechanism. The st ructural and functional consequences of a glutamate to glutamine substituti on in the active site of stromelysin-1 were investigated in this study, In contrast to the wild-type enzyme, the glutamine-substituted mutant was not active in a zymogram assay where gelatin was the substrate, was not activat ed by organomercurials and showed no activity against a peptide substrate. The glutamine-substituted mutant did, however, bind to TIMP-1, the tissue i nhibitor of metalloproteinases, after cleavage of the propeptide with tryps in, A second construct containing the glutamine substitution but lacking th e propeptide was also inactive in the proteolysis assays and capable of TIM P-1 binding. X-ray structures of the wild-type and mutant proteins complexe d with the propeptide-based inhibitor Ro-26-2812 were solved and in both st ructures the inhibitor binds in an orientation the reverse of that of the p ropeptide in the pro-form of the enzyme. The inhibitor makes no specific in teractions with the active site glutamate and a comparison of the wildtype and mutant structures revealed no major structural changes resulting from t he glutamate to glutamine substitution.