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 the activity of its cognate serine protease, coagulation factor VIIa (FVIIa), has been studied using the fluorogenic ester substrate 4-methylumbelliferyl p'-guanidinobenzoate (MUGB). Kinetic data were collected at pH 8.4 and pH 7.6 in the presence and absence of soluble tissue factor (sTF; recombinant human tissue factor containing only the extracellular domain). Pre-steady-state techniques allowed the determination of the individual rate constants for acylation (k2) and deacylation (k3) of the sTF.FVIIa complex as well as the dissociation constant for the noncovalent Michaelis complex with MUGB. Alternative methods were 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. The 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 k2 and k3 were observed. Thus, we conclude that for the ester substrate MUGB, sTF exerts greater allosteric effects on substrate binding than on the later steps involved in the catalytic pathway.
Payne
M A
MA
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Neuenschwander
P F
PF
Johnson
A E
AE
Morrissey
J H
JH
D000494:Allosteric Regulation; D001665:Binding Sites; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D011994:Recombinant Proteins / Q000494:pharmacology; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013925:Thromboplastin / Q000494:pharmacology*
C017532:4-methylumbelliferylguanidinobenzoate; D011994:Recombinant Proteins; D006923:Hymecromone; D013925:Thromboplastin; D015942:Factor VIIa