Mutational analysis of the reactive site loop of Streptomyces metalloproteinase inhibitor, SMPI

Streptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to sh ow "standard mechanism inhibition" against metalloproteinases. SMPI is a gl obular protein with an exposed loop containing the reactive site, C64-V65, To analyze the importance of basic residues in the reactive site loop of SM PI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K 61A, 60/61/66A, and 60/61/66E), The mutants involving only R60, K61, and R6 0/K61 residues, respectively, showed strong inhibitory activity and were st able against enzyme activity. Both the triple mutants showed very weak inhi bitory activity and underwent rapid degradation, The addition of basic resi dues to the loop (V62R and T63R) did not cause any further increase in inhi bitory activity. These results suggest that basic residues in the reactive site loop play some role in maintaining a stable enzyme-inhibitor complex. The R66 mutant showed reduced activity and was rapidly degraded by enzymes, It was concluded that R66 is essential for maintaining a strong hydrophobi c interaction with the S1' hydrophobic pocket of the enzyme, To investigate the roles of the disulfide bridge and the P68 residue near the reactive si te, C64/69S and P68T mutants were constructed. These mutants showed very we ak inhibitory activity and were rapidly degraded by enzymes, These results suggest that the disulfide bridge and P68 residue are very essential for SM PI to function as an inhibitor.