Four loops of the catalytic domain of factor VIIa mediate the effect of the first EGF-like domain substitution on factor VIIa catalytic activity

presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrate d that substitution of the EGF1 domain of factor VIIa with that of factor I X (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinit y and catalytic activity. Supporting simulations of the solution structures of Ca2+-bound factor VIIa and FVII((IXegf1))a with tissue factor are provi ded. Mutants are generated, based on the simulation model, to study the eff ect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for f actor VIIa. The catalytic domain of the chimeric factor VIIa has been distu rbed and several surface loops in the catalytic domain of FVII((IXegf1))a ( Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest large r position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic do main, shifts relative to factor VIIa, resulting in a slight alteration of t he active site. The results suggest that these four loops mediate the effec t of the EGF1 domain substitution on the SI site and catalytic residues. To test the model, we prepared mutations of these surface loops, including fo ur FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A + R147A + D186A) and a FVII mutant with Loop 170s pa rtially deleted, Loop 170s(del). The catalytic activities towards a small p eptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, act ivated), L144Aa and D186Aa, respectively, while MM3a lost almost all cataly tic activity. The combined results of the simulations and mutants provide i nsight into the mechanism by which tissue factor enhances factor VIIa catal ytic activity. (C) 2001 Academic Press.