Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor)

Unlike trypsin-like serine proteases having only one conspicuousbinding pocket in the active site, subtilisin BPN' has two suchpockets, the S1 and S4 pockets, which accommodate the P1 andP4 residues of ligands (after Schechter and Berger notation)respectively. Using computer graphics, the geometrical natureof the two pockets was carefully examined and strategies forsite-directed mutagenesis studies were set up against a proteinSSI (Streptomyces subtilisin inhibitor), which is a strong proteinaceousinhibitor (or a substrate analogue) of subtilisin BPN'. It wasdecided to convert the P1 residue, methionine 73, into lysine(M73K) with or without additional conversion of the P4 residue,methionine 70, into glycine (M70G). The crystal structures ofthe two complexes of subtilisin BPN', one with the single mutantSSI (M73K) and the other with the double mutant SSI (M73K, M70G)were solved showing that (i) small ‘electrostatic induced-fitmovement’ occurs in the S1 pocket upon introducing theterminal plus charge of the lysine side chain, and (ii) large‘mechanical induced-fit movement’ occurs in theS4 pocket upon reducing the size of the P4 side chain from methionineto glycine. In both (i) and (ii), the induced-fit movement occurredin a concerted fashion involving both the enzyme and ‘substrate’amino acid residues. The term ‘substrate-assisted stabilization’was coined to stress the cooperative nature of the induced-fitmovements.