IDENTIFICATION OF REACTIVE-SITE OF A PROTEINACEOUS METALLOPROTEINASE INHIBITOR FROM STREPTOMYCES-NIGRESCENS TK-23

Streptomyces metalloproteinase inhibitor (SMPI), isolated from Strepto myces nigrescens TK-23, is a small proteinaceous metalloproteinase inh ibitor consisting of 102 amino acid residues and two disulfide bridges . SMPI specifically inhibits metalloproteinases such as thermolysin. A fter prolonged incubation with a catalytic amount of thermolysin, it i s cleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., a nd Murao, St (1985) J. Biochem. 97, 173-180]. Hence, for identificatio n of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for inhibitory activity. Among the m, V65I, V65L, V65F, and V65Y retained strong inhibitory activity, whe reas V65S, V65C;, V65E, and V65E showed very weak inhibitory activity against. thermolysin. The Xi values were found to be of the order of 1 0(-10) M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A(2)p r(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzed by means of limited proteolysis with thermolysin. Mutants which retained strong inhibitory activity were cleaved by thermolysin only at the reactive site, in the same way as native SMPI. Tile mutan ts which showed weak inhibitory activity underwent rapid degradation. These results were consistent with the substrate specificity of thermo lysin. Based on these results, the reactive site of SMPI was identifie d as Cys64-Val65.