The hydrophobic S 1 0 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 0 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 0 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 0 position. Rather unexpectedly, the Leu202 !Phe and Leu202 !Tyr mutations, which were expected to decrease the size of the S 1 0 subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P 1 0 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 0 subsite. Thermolysin and TLP-ste variants with identical S 1 0 subsites were highly similar in terms of their preference for Phe vs. Leu in the P 1 0 position.Keywords: metalloendopeptidase; thermolysin; Bacillus stearothermophilus; substrate specificity; hydrophobic binding pocket. (Fig. 1). Four substrate binding pockets (S 2 , S 1 , S 1 0 and S 2 0 ; nomenclature according to Schechter and Berger [6]) have been identified [7]. The S 1 0 subsite is a hydrophobic pocket that is considered to be a major determinant of substrate specificity [8,9]. In thermolysin and the TLP produced by B. stearothermophilus, the subjects of this study, the S 1 0 subsite is mainly formed by Phe130, Val139, Leu202 and Phe133 (TLP-ste) or Leu133 (TLN).Crystallographic [2,7,10,11] and modelling studies [7] of thermolysin have indicated that the S 1 0 subsite allows efficient binding of a leucine side chain. The notion that the S 1 0 subsite in thermolysin is not optimal for binding larger residues, such as phenylalanine [7,11], was experimentally confirmed by Izquierdo and Stein [12]. These authors showed a clear positive correlation between the size of the P 1 0 residue and the activity of the enzyme on dipeptide substrates of the 3-(2-furylacryloyl)-L-glycyl-L-X-amide type (FaGXa, where X is a hydrophobic amino acid). Phenylalanine, however, did not conform to this trend as illustrated by the fact that similar k cat /K m values were obtained for FaGLa and FaGFa. The S 1 0 subsite of TLP-ste is similar in structure and character to that of thermolysin, but TLP-ste has a higher preference for substrates with a Phe at P 1 0 . In the present study, we have investigated the possibility of modifying the S 1 0 subsite in TLPs in order to change the preference of the enzyme for Leu and Phe in the P 1 0 position. Our hypothesis is that a limited increase in the size of the S...