Hydrolysis of small peptide substrates parallels binding of chymotrypsin inhibitor 2 for mutants of subtilisin BPN'.

Variants of subtilisin BPN' that possess improved specificity towards isoleucine compared with alanine at the P4 position of small peptide substrates, were analysed for their ability to bind chymotrypsin inhibitor 2. The binding of the inhibitor with isoleucine (wild-type) and with alanine as the P4 residue parallels the hydrolysis of tetrapeptide substrates. There is a linear relationship between the free energy of binding of the transition state of the substrate and the free energy of binding of the inhibitor with a slope of 2.0. The data suggest that the inhibitor uses predominantly ground state rather than transition state binding energy.