Tissue factor alters the pK_a values of catalytically important factor VIIa residues

Blood coagulation is triggered when the serine protease factor VIIa (fVIIa) binds to cell surface tissue factor (TF) to form the active enzyme-cofactor complex. TF binding to fVIIa allosterically augments the enzymatic activity of fVIIa toward macromolecular substrates and small peptidyl substrates. The mechanism of this enhancement remains unclear. Our previous studies have indicated that soluble TF (sTF; residues 1-219) alters the pH dependence of fVIIa amidolytic activity (Neuenschwander et al. (1993) Thromb. Haemostasis 70, 970), indicating an effect of TF on critical ionizations within the fVIIa active center. The pKa values and identities of these ionizable groups are unknown. To gain additional insight into this effect, we have performed a detailed study of the pH dependence of fVIIa amidolytic activity. Kinetic constants of Chromozym t-PA (MeSO₂-D-Phe-Gly-Arg-pNA) hydrolysis at various pH values were determined for fVIIa alone and in complex with sTF. The pH dependence of both enzymes was adequately represented using a diprotic model. For fVIIa alone, two ionizations were observed in the free enzyme (pK_E1 = 7.46 and pK_E2 = 8.67), with at least a single ionization apparent in the Michaelis complex (pK_ES1 ∼ 7.62). For the fVIIa-sTF complex, the pK_a of one of the two important ionizations in the free enzyme was shifted to a more basic value (pK_E1 = 7.57 and pK_E2 = 9.27), and the ionization in the Michaelis complex was possibly shifted to a more acidic pH (pK_ES1 = 6.93). When these results are compared to those obtained for other well-studied serine proteases, K_E1 and K_ES1 are presumed to represent the ionization of the overall catalytic triad in the absence and presence of substrate, respectively, while K_E2 is presumed to represent ionization of the α-amino group of Ile₁₅₃. Taken together, these results would suggest that sTF binding to fVIIa alters the chemical environment of the fVIIa active site by protecting Ile₁₅₃ from deprotonation in the free enzyme while deprotecting the catalytic triad as a whole when in the Michaelis complex.