Profactor IX propeptide and glutamate substrate binding sites on the vitamin K-dependent carboxylase identified by site-directed mutagenesis

Abstract

The vitamin K-dependent carboxylase, a constituent of the endoplasmic reticulum membrane, catalyzes the conversion of reduced vitamin K to vitamin K epoxide and the concomitant conversion of glutamic acid to γ- carboxyglutamic acid. To study structure-function relationships in the enzyme, seventeen clusters of charged residues of the bovine γ-glutamyl carboxylase were substituted with alanines using site-specific mutagenesis. Wild-type and mutant carboxylase species were expressed in Chinese hamster ovary cells with an immunodetectable octapeptide inserted at their amino- terminal ends. Out of 17 mutant carboxylase species that contain a total of 41 charged residue to alanine substitutions, K217A/K218A (CBX217/218), R234A/H235A (CBX234/235), R359A/H360A/K361A (CBX359/360/361), R406A/H408A (CBX406/408), and R513A/K515A (CBX513/515) had impaired carboxylase activity compared with the wild-type enzyme. The vitamin K epoxidase activities of these mutants were reduced in parallel with the carboxylase activities. CBX217/218 appears to be inactive. High propeptide concentrations were required for stimulation of carboxylation of FLEEL by CBX234/235, CBX406/408, and CBX513/515, suggesting defects in the propeptide binding site. CBX359/360/361 showed normal affinity for the propeptide, FLEEL, proPT28, and vitamin K hydroquinone but exhibited a low catalytic rate for carboxylation. These results suggest that residue 217, residue 218, or both are either critical for catalysis or for maintaining the structure of a catalytically active enzyme. Regions around residues 234, 406, and 513 define in part the propeptide binding site, while the regions around residue 359 are involved in catalysis.