The effect of changing the hydrophobic S-1 ' subsite of thermolysin-like proteases on substrate specificity

The hydrophobic S-1' subsite is one of the major determinants of the substr ate specificity of thermolysin and related M4 family proteases. In the ther molysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-st e), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residu es. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resul ted in a higher efficiency towards substrates with Phe in the P-1' position . Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which w ere expected to decrease the size of the S-1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P-1' posi tion. This is probably due to the fact that 202Phe and 202Tyr adopt a secon d possible rotamer that opens up the subsite compared to Leu202, and also f avours interactions with the substrate. To validate these results, we const ructed variants of thermolysin with changes in the S-1' subsite. Thermolysi n and TLP-ste variants with identical S-1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P-1' position.