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 established. Earlier studies of the pH dependence of factor XIII activity suggested the existence...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-03, Vol.269 (12), p.9190-9194
Main Authors: Micanovic, R, Procyk, R, Lin, W, Matsueda, G R
Format: Article
Language:English
Subjects:
Binding Sites
Biological and medical sciences
Blood coagulation. Blood cells
Catalysis
Coagulation factors
Factor XIII - chemistry
Factor XIII - metabolism
Fundamental and applied biological sciences. Psychology
Gene Expression
Histidine - chemistry
Humans
Molecular and cellular biology
Mutagenesis, Site-Directed
RNA, Messenger - genetics
Structure-Activity Relationship
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Summary: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 established. Earlier studies of the pH dependence of factor XIII activity suggested the existence of a putative active site histidine. We used chemical modification and oligonucleotide directed site-specific mutagenesis to investigate the role of histidines. Photo-oxidation with methylene blue resulted in a complete loss of catalytic activity under conditions that oxidized histidine but did not affect the essential cysteine. Single substitution of each of the 14 histidine residues in the a-subunit of factor XIII by asparagine or alanine led to mutants with catalytic 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 [14C]putrescine incorporation into dimethylcasein, and failed to cross-link fibrin gamma-chains. Thus, the a-subunit His-373 may function in the active site of factor XIII, by analogy with papain's mechanism, as a histidinium cation that increases the nucleophilicity of the essential Cys-314. Decreased expression levels of His-373 mutants also indicate that this residue may be critical for enzyme stability.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)37093-X