Open AccessThe effect of changing the hydrophobic S 1 ′ subsite of thermolysin‐like proteases on substrate specificity
Author(s) -
de Kreij Arno,
van den Burg Bertus,
Veltman Oene R.,
Vriend Gert,
Venema Gerard,
Eijsink Vincent G. H.
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.0014-2956.2001.02434.x
Subject(s) - thermolysin , dipeptide , chemistry , proteases , thermostability , stereochemistry , substrate (aquarium) , protease , biochemistry , amino acid , enzyme , biology , trypsin , ecology
The hydrophobic S 1 ′ subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin‐like protease (TLP) produced by Bacillus stearothermophilus (TLP‐ste), the hydrophobic S 1 ′ 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 residues. The mutational effects showed that the wild‐type S 1 ′ pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P 1 ′ position. Rather unexpectedly, the Leu202→Phe and Leu202→Tyr mutations, which were 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 ′ position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S 1 ′ subsite. Thermolysin 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.