文摘
Serine proteases of the chymotrypsin family show a dichotomous amino acid distribution forresidue 225. Enzymes carrying Tyr at position 225 are activated by Na+, whereas those carrying Pro aredevoid of Na+ binding and activation. Previous studies have demonstrated that the Y225P conversion issufficient to abrogate Na+ activation in several enzymes. However, the reverse substitution P225Y isnecessary but not sufficient to introduce Na+ binding and activation. Here we report that Streptomycesgriseus trypsin, carrying Pro-225, can be engineered into a Na+-activated enzyme by replacing residuesin the 170, 186, and 220 loops to those of coagulation factor Xa. The findings represent the first instanceof an engineered Na+-activated enzyme and a proof of principle that should enable the design of otherproteases with enhanced catalytic activity and allosteric regulation mediated by monovalent cation binding.
