Identification of Reactive Site of a Proteinaceous MetalloproteinaseInhibitor from Streptomyces nigrescens TK-23

Abstract

Streptomyces metalloproteinase inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous metalloproteinase inhibitor consisting of 102 amino acidresidues and two disulfide bridges. SMPI specifically inhibits metalloproteinases such asthermolysin. After prolonged incubation with a catalytic amount of thermolysin, it iscleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification 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 them, V65I, V65L, V65F, and V65Y retained strong inhibitory activity, whereas V65S, V65G, V65K, and V65Eshowed very weak inhibitory activity against thermolysin. The K₁ values were found to beof the order of 10^-10M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A₂pr(Dnp)-Ala-Arg-NH₂. In addition, susceptibility to enzyme degradation was analyzedby means of limited proteolysis with thermolysin. Mutants which retained strong inhibi-tory activity were cleaved by thermolysin only at the reactive site, in the same way asnative SMPI. The mutants which showed weak inhibitory activity underwent rapiddegradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.