COAGULATION-FACTOR XIIIA UNDERGOES A CONFORMATIONAL CHANGE EVOKED BY GLUTAMINE SUBSTRATE - STUDIES ON KINETICS OF INHIBITION AND BINDING OFXIIIA BY A CROSS-REACTING ANTIFIBRINOGEN ANTIBODY

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamin e:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond fo rmation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen A a lpha-chain segment 529-539 was previously observed from analysis of en d-stage plasma clots to block fibrin or-chain cross-linking. This prom pted the study of its effect on nonfibrinogen substrates, with the pro spect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catal yzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect t o dansylcadaverine, Uncompetitive inhibition was also observed with th e synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetit ive inhibition arises from preferential interaction of the inhibitor w ith the enzyme-substrate complex but is also found to inhibit gamma-ch ain cross-linking. The conjunction of the uncompetitive and competitiv e modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substra te precedes the amine exchange. The presence of substrate enhanced bin ding of 5A2 to XIIIa, an interaction deemed to occur through a C-termi nal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of w hich comprises sequences occurring in the fibrinogen epitope A alpha-( 529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa a bolishes the inhibitory effect of 5A2 on activity. Crystallographic st udies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site a nd have predicted that for catalysis, a conformational change may acco mpany glutamine-substrate binding. The uncompetitive inhibition and th e substrate-dependent binding of 5A2 provide evidence for the conforma tional change.