PROBING CATALYTIC HINGE BENDING MOTIONS IN THERMOLYSIN-LIKE PROTEASESBY GLYCINE-]ALANINE MUTATIONS

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallogra phic studies have shown that the active-site cleft is more closed in L igand-binding TLPs than in Ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis res ulting in ''closure'' and ''opening'' of the active-site cleft. Two hi nge regions have been proposed. One is located around a conserved glyc ine 78; the second involves residues 135 and 136. The importance of co nserved glycine residues in these hinge regions was studied experiment ally by analyzing the effects of Gly --> Ala mutations on catalytic ac tivity. Eight such mutations were made in the TLP of Bacillus stearoth ermophilus (TLP-ste) and their effects on activity toward casein and v arious peptide substrates were determined. Only the Gly78Ala, Gly136Al a, and Gly135Ala + Gly136Ala mutants decreased catalytic activity sign ificantly. These mutants displayed a reduction in k(cat)/K-m for 3-(2- furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respecti vely. Comparisons of effects on k(cat)/K-m for various substrates with effects on the K-i for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence k(cat). The app arent importance of conserved glycine residues in proposed hinge-bendi ng regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.