Abstract
A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XIII A-chains (FXIII A), designated as delta K513, delta A502, delta Y481, and delta K462, respectively, were expressed in Escherichia coli to define the minimum structure required for transglutaminase activity. delta K513 and delta A502 displayed a 3.8-4.7-fold reduction in the Kcat with no change in the Km for the glutamine substrate and a 2-fold increase in the Km of the primary amine substrate. There was no detectable transglutaminase activity for either thrombin-activated delta Y481 or delta K462. The rate of ammonia release of thrombin-activated delta K513 and delta A502 was reduced 6- and 4-fold, respectively, whereas ammonia release was not detected for the delta Y481 and delta 462 mutants. The Kact for calcium ions of the delta K513 mutant was similar to recombinant FXIIIa, whereas, it was increased by approximately 3-fold for the delta A502 mutant. The rate of fibrin gamma-chain dimer formation for the delta K513 and delta A502 mutants was reduced by approximately 19-fold. delta K462 did not bind to fibrin, while all of the other thrombin-cleaved mutants were bound. In conclusion, these results documented that the carboxyl-terminal calcium binding domain (Asp468-Glu495) was important for FXIIIa to adopt the correct conformation to ensure that efficient catalysis occurred.
Highlights
A series of truncation mutants lacking 218, 229, 250, the posttranslationalmodification of proteins by forming stable and 269 amino acid residues from the carboxyl terminus y-glutamyl-e-lysyl covalent bonds between proteins [1,2]
The of bloodcoagulation factor XI11 A-chains (FXIIIA), des- transglutaminases have a high degree of amino acid sequence ignated as AK513A, A502A, Y481, and AK462, respec- homology demonstrating that these enzymes share a common tively, were expressed in Escherichia coli to define the ancestry [4]
The rate of ammoniarelease of throm- In plasma, blood coagulation FXIII is a tetrameric zymogen bin-activated AK513 and a 5 0 2 was reduced 6- and composed of two A-chains(81kDa) andtwo B-chains (75 kDa), 4-fold, respectively, whereas ammonia release was not whereas monocytes and platelet factor XI11 are dimeric comdetected for the AY481 and A462 mutants
Summary
There are other transglutaminases, including the keratinocytetransglutaminase,tissuetransglutaminase, or epidermal transglutaminase, and prostate transglutaminase [1,2], involved in diverse physiological processes that catalyze while others reported thata catalytically active fibrin binding fragment was isolated whencalcium ions were present during proteolysis [8]. These discordant results could be an effect of the putativecalcium binding domain (Thr458-G1~495) locaitend the carboxyl-terminal portion of the Gly38-Lys513fragment in regulating FXIIIaactivity [7]. Detailedkineticanalysis of the biochemical properties of the Gly3*-Lys513fragment and additional carboxyl-terminal deletions could further define the role of the putative calciumbinding domain in FXIIIafunction
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