EFFECT OF SOLUBLE TISSUE FACTOR ON THE KINETIC MECHANISM OF FACTOR VIIA - ENHANCEMENT OF P-GUANIDINOBENZOATE SUBSTRATE HYDROLYSIS

The mechanism by which the protein cofactor, tissue factor, enhances t he activity of its cognate serine protease, coagulation factor VIIa (F VIIa), has been studied using the fluorogenic ester substrate 4-methyl umbelliferyl p'-guanidinobenzoate (MUGB). Kinetic data were collected at pH 8.4 and pH 7.6 in the presence and absence of soluble tissue fac tor (sTF; recombinant human tissue factor containing only the extracel lular domain). Pre-steady-state techniques allowed the determination o f the individual rate constants for acylation (k(2)) and deacylation ( k(3)) of the sTF . FVIIa complex as well as the dissociation constant for the noncovalent Michaelis complex with MUGB. Alternative methods w ere required for determination of these parameters for free FVIIa due to extremely slow hydrolysis of MUGB in the absence of sTF. Under all experimental conditions, deacylation was found to be rate-limiting. Th e major effect of sTF was to raise the affinity of FVIIa for MUGB (31- fold at pH 8.4 and 36-fold at pH 7.6); only minor changes in k(2) and k(3) were observed. Thus, we conclude that for the ester substrate MUG B, sTF exerts greater allosteric effects on substrate binding than on the later steps involved in the catalytic pathway.