Unexpected crucial role of residue 225 in serine proteases

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively foun d in vertebrates, whereas proteases with P225 (like degradative enzymes) ar e present from bacteria to human. Saturation mutagenesis of Y225 in thrombi n shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structur es of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primar y specificity site. The activity profiles obtained for thrombin also sugges t that the conversion of Pro to Tyr or Phe documented in the vertebrates oc curred through Ser and aas driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine pro teases, which together with Pro and Tyr account for almost the entire distr ibution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activ ity and specificity are more complex than currently believed. These finding s have broad implications in the rational design of enzymes with enhanced c atalytic properties.