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 ␥-carboxyglutamic acid. To study structure-function relationships in the enzyme, seventeen clusters of charged residues of the bovine ␥-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.Vitamin K-dependent ␥-glutamyl carboxylase is a membrane-associated endoplasmic reticulum resident enzyme that catalyzes the posttranslational conversion of specific glutamic acids of the vitamin K-dependent proteins to Gla. The vitamin K-dependent blood-clotting and regulatory proteins have clusters of 9 -12 Gla residues near their N termini. A Ca 2ϩ

A Conserved Region of Human Vitamin K-dependent Carboxylase between Residues 393 and 404 Is Important for Its Interaction with the Glutamate Substrate

“…Therefore, all kinetic studies of the W399A mutant were performed at 28 M KH 2 . The reason for onset of inhibition at a lower KH 2 concentration for W399A is not clear, but similar phenomena have been reported in some other carboxylase mutants (31). The rate of carboxylation measured at 28 M KH 2 for W399A is about 70% of the V max predicted from extrapolating the initial non-inhibitory phase of the kinetic curve.…”

“…lower vitamin K concentrations (31). W399A is inhibited at lower vitamin K concentrations than either wild type or the other mutants we have studied.…”

Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state

“…Construction of the FLAG-carboxylase cDNA and expression of the recombinant enzyme in Chinese hamster ovary (CHO) cells has been described (22). A clone of CHO cells stably expressing the FLAG-carboxylase was subjected to stepwise selection with methotrexate to amplify expression of the enzyme.…”

“…Construction of the expression vector for FLAG-carboxylase, transfection of CHO cells, and expression of FLAGcarboxylase have been described (22). Cells from a stable colony producing the highest level of enzyme were selected stepwise with increasing concentrations of methotrexate, up to 0.15 M, to augment carboxylase production.…”

“…Bound antibodies were detected with the ECL detection kit (Amersham) followed by autoradiography on Hyperfilm ECL (Amersham). Quantitation of FLAG-carboxylase concentration was performed as described (22).…”