ROLE OF HISTIDINE-373 IN THE CATALYTIC ACTIVITY OF COAGULATION-FACTOR-XIII

Factor XIII catalysis proceeds via formation of thioester acyl enzyme intermediate involving an active site cysteine residue at position 314 . The contribution of other residues to catalysis has not been establi shed. Earlier studies of the pH dependence of factor XIII activity sug gested the existence of a putative active site histidine. We used chem ical modification and oligonucle otide directed site-specific mutagene sis to investigate the role of histidines. Photo-oxidation with methyl ene blue resulted in a complete loss of catalytic activity under condi tions that oxidized histidine but did not affect the essential cystein e. Single substitution of each of the 14 histidine residues in the a s ubunit of factor XIII by asparagine or alanine led to mutants with cat alytic activities generally not significantly different from the wild- type recombinant enzyme. The only exceptions were the H373N and H373A mutants that were poorly expressed, had no detectable rate of [C-14]pu trescine in corporation into dimethylcasein, and failed to cross-link fibrin gamma-chains. Thus, the a subunit His-373 may function in the a ctive site of factor XIII, by analogy with papain's mechanism, as a hi stidinium cation that increases the nucleophilicity of the essential C ys-314. Decreased expression levels of His-373 mutants also indicate t hat this residue may be critical for enzyme stability.