A conserved region of human vitamin K-dependent carboxylase between residues 393 and 404 is important for its interaction with the glutamate substrate.
Certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their gamma-glutamyl carboxylase causing impaired glutamate binding. The sequence surrounding Leu394 is similar in all known carboxylases, suggesting that the region is functionally important. To test this hypothesis we made the following mutant enzymes: W390A, Y395A, S398A, W399A, and H404A. We purified the enzymes and corrected the activity measurements for active enzyme concentration. Carboxylases W390A, S398A, and H404A had activities similar to that of wild type; however, Y395A and W399A had lower activities than did wild type. In the following descriptions we include our previously reported results for L394R. Kinetic studies with the substrate FLEEL, revealed Km values of 0.5 (wild type), 6.5 (L394R), 15 (Y395A), and 24 (W399A) mm. The kcat values relative to wild type were 51% (L394R), 1% (Y395A), and 2% (W399A). The kcat/Km values were 24-fold (L394R) and >2000-fold lower for Y395A and W399A than for wild-type carboxylase. Inhibition of FLEEL carboxylation by the competitive inhibitor, Boc-mEEV, gave Ki values of 0.013 (wild type), 1.4 (L394R), 2.1 (Y395A), and >5 (W399A) mm. The Y395A propeptide affinity was similar to that of wild type, but those of L394R and W399A were 16-22-fold less than that of wild type. Results of kinetic studies with a propeptide-containing substrate were consistent with results of propeptide binding and FLEEL kinetics. Although propeptide and vitamin K binding in some mutants were affected, our data provide compelling evidence that glutamate recognition is the primary function of the conserved region around Leu394.
Mutucumarana
Vasantha P
VP
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.
Acher
Francine
F
Straight
David L
DL
Jin
Da-Yun
DY
Stafford
Darrel W
DW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001667:Binding, Competitive; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics
D018076:DNA, Complementary; D009842:Oligopeptides; C040586:phenylalanyl-leucyl-glutamyl-glutamyl-isoleucine; D018698:Glutamic Acid; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase