Carboxyl-terminal truncation of recombinant factor XIII A-chains. Characterization of minimum structural requirement for transglutaminase activity.
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.
Lai
T S
TS
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Achyuthan
K E
KE
Santiago
M A
MA
Greenberg
G S
GS
D001483:Base Sequence; D001665:Binding Sites; D005337:Fibrin / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D011503:Transglutaminases / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D005337:Fibrin; D011503:Transglutaminases