MUTATIONAL REPLACEMENTS OF THE AMINO-ACID-RESIDUES FORMING THE HYDROPHOBIC S(4) BINDING POCKET OF SUBTILISIN-309 FROM BACILLUS-LENTUS

The amino acid side chains of Ile107, Leu126, and Leu 135 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 k(cat)t/K(M) 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. Ile107 provides important van der Waals contacts with aromatic as well as aliphatic side chains, but some steric repulsion in the case of branched aliphatic P4 side chains, in particular with Leu, complicates the picture. Some of this strain is alleviated by the Ile107 --> Gly, Ala, or Val substitution. All the mutants, including those with the most sterically restricted S4 sites, hydrolyze an optimized substrate (very high k(cat)/K(M)) with a Phe at the P4 position at rates varying only 2-3-fold as compared to 84-fold for a nonoptimized substrate (low k(cat)/K(M)). These results suggest that the availability of sufficient overall binding energy can overcome the steric repulsion at a single subsite.