Exosite interactions determine the affinity of factor X for the extrinsic Xase complex

The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, fact or VIIa, bound to tissue factor (TF) on phospholipid membranes. We have inv estigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VII a within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X, This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical nonc ompetitive inhibition of factor X activation by active site-directed ligand s. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protei n substrate affinity, whereas the second binding step influences the maximu m catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivat ives that are expected to have limited or abrogated interactions with the a ctive site of VIIa within Xase. Thus, substrate interactions at exosites, s ites removed from the active site of VIIa within the enzyme complex, determ ine affinity and binding specificity in the productive recognition of facto r X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homo logous enzymes of coagulation.