Effect on inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficientlyinhibits a bacterial serine protease, subtilisin BPN'. We recentlydemonstrated that functional change in SSI was possible simplyby replacing the amino acid residue at the reactive P1 site(methionine 73) of SSI. The present paper reports the additionaleffect of replacing methionine 70 at the P4 site of SSI(Lys73)on inhibitory activity toward two types of serine proteases,trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversionof methionine 70 at the P4 site of SSI(Lys73) to glycine oralanine resulted in increased inhibitory activity toward trypsinand lysyl endopeptidase, while replacement with phenylalanineweakened the inhibitory activity toward trypsin. This suggeststhat steric hindrance at the P4 site of SSI(Lys73) is an obstaclefor its binding with trypsin. In contrast, the same P4 replacementshad hardly any effect on inhibitory activity toward subtilisinBPN'. Thus the subsite structure of subtilisin BPN' is tolerantto these replacements. This contrast in the effect of P4 substitutionmight be due to the differences in the S4 subsite structuresbetween the trypsin-like and the subtilisin-like proteases.These findings demonstrate the importance of considering structuralcomplementarity, not only at the main reactive site but alsoat subsites of a protease, when designing stronger inhibitors.