Abstract
Coagulation Factor VIIa (FVIIa) lacks the ability to spontaneously complete the conversion to a fully active enzyme after specific cleavage of an internal peptide bond (Arg152-Ile153) in the zymogen. Recently, several variants of FVIIa with enhanced intrinsic activity have been constructed. The in vitro characterization of these variants has shed light on molecular determinants that put restrictions on FVIIa in favour of a zymogen-like conformation and warrants continued efforts. Here we describe a new FVIIa variant with high intrinsic activity containing the mutations Leu305-->Val, Ser314-->Glu, Lys337-->Ala, and Phe374-->Tyr. The variant, called FVIIa(VEAY), processes a tripeptidyl substrate very efficiently because of an unprecedented, 5.5-fold lowering of the K(m) value. Together with a 4-fold higher substrate turnover rate this gives the variant a catalytic efficiency 22 times that of wild-type FVIIa, which is reflected in a considerably enhanced susceptibility to inhibition by antithrombin and other inhibitors. For instance, the affinity of FVIIa(VEAY) for the S1 probe and inhibitor p -aminobenzamidine is represented by an 8-fold lower K(i) value compared with that of FVIIa. Activation of Factor X in solution occurs about 10 times faster with FVIIa(VEAY) than with FVIIa, due virtually exclusively to an increased kcat value. The high activity of FVIIa(VEAY) is not accompanied by an increased burial of the N-terminus of the protease domain. A comparison of the kinetic parameters and molecular properties of FVIIa(VEAY) with those of the previously described mutant V158D/E296V/M298Q-FVIIa (FVIIa(IIa)), and the locations of the substitutions in the two variants, reveals what appear to be two profoundly different structural mechanisms dictating improvements in enzymic performance.