Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody

• Published in: The Journal of biological chemistry 1998-06, Vol.273 (23), p.14387-14391
• Main Authors: Mitkevich, O V, Shainoff, J R, DiBello, P M, Yee, V C, Teller, D C, Smejkal, G B, Bishop, P D, Kolotushkina, I S, Fickenscher, K, Samokhin, G P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - pharmacology; Cadaverine - analogs & derivatives; Cadaverine - metabolism; Enzyme Inhibitors - immunology; Fibrinogen - immunology; Glutamine - metabolism; Humans; Kinetics; Models, Molecular; Molecular Sequence Data; Peptide Fragments - metabolism; Peptides - pharmacology; Protein Binding - physiology; Protein Conformation; Transglutaminases - chemistry; Transglutaminases - immunology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.273.23.14387

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed 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 to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.