PREFACTOR IX PROPEPTIDE AND GLUTAMATE SUBSTRATE-BINDING SITES ON THE VITAMIN-K-DEPENDENT CARBOXYLASE IDENTIFIED BY SITE-DIRECTED MUTAGENESIS

The vitamin K-dependent carboxylase, a constituent of the endoplasmic reticulum membrane, catalyzes the conversion of reduced vitamin K to v itamin K epoxide and the concomitant conversion of glutamic acid to ga mma-carboxyglutamic acid, To study structure-function relationships in the enzyme, seventeen clusters of charged residues of the bovine gamm a-glutamyl carboxylase were substituted with alanines using site-speci fic mutagenesis, Wild-type and mutant carboxylase species were express ed in Chinese hamster ovary cells with an immunodetectable octapeptide inserted at their amino-terminal ends, Out of 17 mutant carboxylase s pecies that contain a total of 41 charged residue to alanine substitut ions, K217A/K218A (CBX217/218), R234A/H235A (CBX234/235), R359A/H360A/ K361A. (CBX359/360/361), R406A/H408A (CBX406/408), and R513A/K515A (CE X513/515) had impaired carboxylase activity compared with the wild-typ e enzyme, The vitamin K epoxidase activities of these mutants were red uced in parallel with the carboxylase activities, CBX217/218 appears t o be inactive, High propeptide concentrations were required for stimul ation 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 vitami n K hydroquinone but exhibited a low catalytic rate for carboxylation. These results suggest that residue 217, residue 218, or both are eith er critical for catalysis or for maintaining the structure of a cataly tically active enzyme, Regions around residues 234, 406, and 513 defin e in part the propeptide binding site, while the regions around residu e 359 are involved in catalysis.