Bacillus licheniformis variant DY proteinase: specificity in relation to the geometry of the substrate recognition site

Abstract

S1-S4 specificity of the Bacillus licheniformis variant DY proteinase (subtilisin DY) was determined by a series of peptide nitroanilides. The broad S1 specificity is due to the relative flexibility of the binding loop, which exhibits a preference for phenylalanine and accepts poorly the side chains of alanine, valine, lysine, and especially that of glutamic acid, due probably to a steric repulsion by Asn 155 and the narrow entrance of the "pocket." Alanine in position P2 of the substrate is more favorable for the catalysis than glycine. S3 is located on the protein surface. It is more open than the other subsites and can accept a variety of residues. S4 exhibits an extremely high affinity for the aromatic group of phenylalanine. Evidently, hydrophobic forces predominate in the S4--P4 interactions. The results characterize subtilisin DY as a bacterial proteinase with a broad specificity due to the specific geometry and flexibility of the substrate recognition site, which can accommodate different types of amino acid side chains.