Macromolecular substrate-binding exosites on both the heavy and light chains of factor XIa mediate the formation of the Michaelis complex required for factor IX-activation.
Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). To gain further insight into the mechanisms of activation of FIX by FXIa, we have investigated the kinetic properties of FXIa-light chain (FXIa-LC) with its active site occupied by either a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB) or a small peptidyl substrate (S-2366) and have examined FIX cleavage products resulting from activation by FXIa or FXIa-LC. PAB inhibited the hydrolysis of S-2366 by FXIa-LC in a classically competitive fashion. In contrast, PAB was found to be a noncompetitive inhibitor of the activation of the macromolecular substrate FIX. Occupancy of the active site of the FXIa-LC by S-2366 also resulted in noncompetitive inhibition of FIX activation. These results demonstrate the presence of an exosite for FIX binding on the FXIa-LC remote from its active site. Furthermore, examination of the cleavage products of FIX indicated that in the absence of either Ca2+ or the heavy chain of FXIa there was substantial accumulation of the inactive intermediate FIXalpha, indicating a slower rate of cleavage of the scissile bond Arg180-Val181. We conclude that binding to two substrate-binding exosites one on the heavy chain and the other on the light chain of FXIa is required to mediate the formation of the Michaelis complex and efficient cleavages of the two spatially separated scissile bonds of FIX.
Sinha
Dipali
D
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. dipali@temple.edu
Marcinkiewicz
Mariola
M
Navaneetham
Duraiswamy
D
Walsh
Peter N
PN
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D020134:Catalytic Domain; D005164:Factor IX / Q000378:metabolism*; D015945:Factor XIa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity
D005164:Factor IX; D012697:Serine Endopeptidases; D015945:Factor XIa; D002118:Calcium