Factor VIIa modified in the 170 loop shows enhanced catalytic activity butdoes not change the zymogen-like property

Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appear s to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is as sumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond bet ween Cys(310) and Cys(329), which is five residues longer than those of oth er trypsin types. To examine the functional role of the loop region, we pre pared two mutants of VIIa, One of the mutants, named VII-11, had five extra corresponding residues 316-320 of VII deleted. The other mutant, VII-31, h ad all of the residues in its loop replaced! with those of trypsin, Functio nal analysis of the two mutants showed that VIIa-11 (K-d = 41 nM) and VIIa- 31 (K-d = 160 nM) had lower affinities for soluble tissue factor as compare d with the wild-type VIIa (K-d = 11 nM). The magnitude of tissue factor-med iated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced ac tivity; the catalytic efficiencies of VIIa-31 toward various chromogenic su bstrates were 2-18-fold greater than those off the wildtype VIIa. Susceptib ility of the alpha -amino group of IIe-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wildtype VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational tran sition, and changed the catalytic efficiency of VIIa, but they did not affe ct the equilibrium between active and zymogen-like conformational states.