Hiraga K, Seeram S S, Tate S, Tanaka N, Kainosho M, Oda K
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, 606-8585, Japan.
J Biochem. 1999 Jan;125(1):202-9. doi: 10.1093/oxfordjournals.jbchem.a022260.
Streptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to show "standard mechanism inhibition" against metalloproteinases. SMPI is a globular protein with an exposed loop containing the reactive site, C64-V65. To analyze the importance of basic residues in the reactive site loop of SMPI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K61A, 60/61/66A, and 60/61/66E). The mutants involving only R60, K61, and R60/K61 residues, respectively, showed strong inhibitory activity and were stable against enzyme activity. Both the triple mutants showed very weak inhibitory activity and underwent rapid degradation. The addition of basic residues to the loop (V62R and T63R) did not cause any further increase in inhibitory 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 hydrophobic interaction with the S1' hydrophobic pocket of the enzyme. To investigate the roles of the disulfide bridge and the P68 residue near the reactive site, C64/69S and P68T mutants were constructed. These mutants showed very weak inhibitory activity and were rapidly degraded by enzymes. These results suggest that the disulfide bridge and P68 residue are very essential for SMPI to function as an inhibitor.
链霉菌金属蛋白酶抑制剂（SMPI）是唯一一种对金属蛋白酶表现出“标准机制抑制”的抑制剂。SMPI是一种球状蛋白，有一个包含反应位点C64-V65的暴露环。为了分析SMPI反应位点环中碱性残基的重要性，构建了R60、K61和R66的突变体（R60A、K61A、R66A、R60/K61A、60/61/66A和60/61/66E）。分别仅涉及R60、K61和R60/K61残基的突变体表现出很强的抑制活性，并且对酶活性稳定。这两种三突变体均表现出非常弱的抑制活性，并迅速降解。向环中添加碱性残基（V62R和T63R）并未导致抑制活性进一步增加。这些结果表明，反应位点环中的碱性残基在维持稳定的酶-抑制剂复合物中发挥了一定作用。R66突变体活性降低，并被酶迅速降解。得出的结论是，R66对于与酶的S1'疏水口袋保持强烈的疏水相互作用至关重要。为了研究反应位点附近二硫键和P68残基的作用，构建了C64/69S和P68T突变体。这些突变体表现出非常弱的抑制活性，并被酶迅速降解。这些结果表明，二硫键和P68残基对于SMPI作为抑制剂发挥功能非常重要。
