benzoyl-ile-glu-gly-arg-p-nitroanilide and dansylglutamyl-glycyl-arginine-chloromethyl-ketone

The effect of divalent and monovalent cations on the hydrolysis of BzIleGlu(OR)GlyArgpNA(S-2222) was compared to the rate of inactivation of factor Xa by dansyl-GluGlyArg-chloromethylketone(DERG-CK). At substrate concentrations below Km, an approximate four-fold increase in amidase activity was observed in the presence of manganese ions while a three-fold increase was observed with calcium ions. The presence of magnesium ions resulted in a two-fold increase in amidase activity. Similar increases in the rate of inactivation of factor Xa by DERG-CK were observed. Na+ ions had a marked enhancing effect of both factor Xa amidase activity and inactivation by DERG-CK. Kinetic parameters for the hydrolysis of S-2222 by factor Xa were obtained in the presence and absence of Ca++ and Na+. Vmax values increased in the presence of either Ca++ or Na+. Km values increased in the presence of Ca++ while there was a modest decrease in Km in the presence of Na+. It is suggested that the enhanced activity of factor Xa is a reflection of changes in the reactivity of active site residues.

Topics: Amino Acid Chloromethyl Ketones; Binding Sites; Calcium; Cations, Divalent; Cations, Monovalent; Dansyl Compounds; Factor Xa; Humans; Hydrolysis; Kinetics; Lithium; Magnesium; Manganese; Oligopeptides; Potassium; Serine Endopeptidases; Serine Proteinase Inhibitors; Sodium