The amino acid side chains of Ile107, Leu126, and Leu135 participate in the formation of the important hydrophobic S4 binding pocket of the subtilisin Savinase. Ile107 and Leu126, located on each side of the pocket, point toward each other, and Leu135 is situated at the bottom of the pocket. These amino acid residues have been substituted for other hydrophobic amino acid residues by site-directed mutagenesis, and the resulting enzymes have been characterized with respect to their P4 substrate preferences. The Leu126-->Ala or Phe substitutions reduce kcat/KM for the hydrolysis of all substrates to around 5% without altering the substrate preference. It is concluded that Leu126 is an essential structural part of the pocket which cannot be replaced without seriously affecting catalysis, consistent with the fact that Leu126 is conserved among all subtilisins. In contrast, the Ile107-->Gly, Ala, Val, Leu, or Phe and Leu135-->Ala, Val, or Phe substitutions strongly influence the P4 substrate preference, and some of the mutants exhibit large specificity changes for particular substrates when compared to wild-type Savinase. The results can be rationalized on the basis of Ile107 and Leu135 being responsible for steric repulsion of branched aliphatic and aromatic P4 side chains, respectively. Leu135 exclusively interacts with aromatic P4 side chains, and its replacement with less bulky amino acid residues alleviates steric repulsion such that the activity toward this type of substrates is enhanced. Conversely, the introduction of a more bulky amino acid residue at position 135 produces more steric repulsion and reduces the activity toward substrates with aromatic P4 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)