Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (

Biochemistry
26
:
3768
–3775,
1987
). We now provide evidence that a second substrate binding exosite resides on the light chain (LC) of FXIa. To understand the mechanism of activation of FIX by LC we have investigated 1) the kinetics of S-2366 hydrolysis by FXIa and its isolated LC in the presence and absence of a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB); 2) the kinetics of activation of the macromolecular substrate FIX by FXIa and its isolated light chain in the presence and absence of either PAB or the alternative substrate S-2366; and 3) the effect of active site inhibited FXIa LC (LCi, i.e., FXIa LC treated with phenylmethylsulfonyl fluoride) on the activation of FIX by FXIa. Kinetic parameters for the hydrolysis of S-2366 by FXIa and its isolated LC are comparable. However kcat for the activation of the macromolecular substrate FIX by LC compared to full-length FXIa is reduced by ~300-fold whereas the Km was only slightly higher (2- to 3-fold). PAB inhibited the hydrolysis of S-2366 both by FXIa and LC in a classical competitive fashion. If an exosite on the heavy chain of FXIa is solely responsible for formation of the Michaelis complex then PAB should have inhibited the activation of FIX by LC in a competitive fashion. However, PAB and S-2366 were found to be noncompetitive inhibitors of FIX activation by FXIa or LC demonstrating the presence of an exosite for FIX binding on the LC remote from its active site. The presence of the second exosite on the light chain of FXIa was further confirmed by the fact that the V vs. S as well as the V vs. I plots of the activation of FIX by FXIa in the presence of LCi were sigmoidal suggesting formation of a nonproductive SI complex. We conclude that for FIX-activation, two macromolecular substrate-binding exosites, one on the heavy chain and one on the light chain of FXIa are required to mediate the formation of the Michaelis complex.

Topics:
antigens, cd98 light chains, factor ix, factor xia, phenylmethylsulfonyl fluoride, serine proteases

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2005, The American Society of Hematology
2005
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