Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in Vitamin K metabolism in rat liver

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

Section: Discussionmentioning

confidence: 99%

Profactor IX Propeptide and Glutamate Substrate Binding Sites on the Vitamin K-dependent Carboxylase Identified by Site-directed Mutagenesis

Sugiura

Furie

Walsh

et al. 1996

“…On the other hand, lysine 218, and cysteines 99 and 450 are predicted to be in the lumen of the endoplasmic reticulum where catalytic activity is expected to reside (48). Since very little is known about the tertiary structure of the carboxylase, the proximity of these regions to the region focused in our study is unknown.…”

Section: Vitamin K-dependent Carboxylase Glutamate Binding Residuesmentioning

confidence: 96%

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

Mutucumarana

Acher

Straight

et al. 2003

Certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their ␥-glutamyl carboxylase causing impaired glutamate binding. The sequence surrounding Leu 394 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 K m values of 0.5 (wild type), 6.5 (L394R), 15 (Y395A), and 24 (W399A) mM. The k cat values relative to wild type were 51% (L394R), 1% (Y395A), and 2% (W399A). The k cat /K m 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 K i 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 Leu 394 .

“…Finally, two ion fragments predicted from peptide 92-100 were observed at m/z 662.3 and 889. 4. (Fig.…”

Section: Peptide Mass Fingerprint Of Human Vitamin K-dependentmentioning

confidence: 99%

“…The carboxylation reaction occurs in the lumen of the ER (3,4) and uses the substrates carbon dioxide, oxygen, and vitamin K hydroquinone. During the process of carboxylation, the ␥-proton of the glutamic acid is abstracted, followed by the addition of carbon dioxide (5).…”

mentioning

confidence: 99%

Determination of Disulfide Bond Assignment of Human Vitamin K-dependent γ-Glutamyl Carboxylase by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Tie¹,

Mutucumarana²,

Straight³

et al. 2003

Vitamin K-dependent ␥-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to ␥-carboxyglutamate. Carboxylase has ten cysteine residues, but their form The vitamin K-dependent carboxylase is an integral membrane glycoprotein that catalyzes the post-translational modification of specific glutamic acid residues to ␥-carboxyglutamic acid (Gla) 1 (1, 2). The carboxylation reaction occurs in the lumen of the ER (3, 4) and uses the substrates carbon dioxide, oxygen, and vitamin K hydroquinone. During the process of carboxylation, the ␥-proton of the glutamic acid is abstracted, followed by the addition of carbon dioxide (5). Simultaneous with carboxylation, the vitamin K hydroquinone is converted to vitamin K epoxide, which is converted back to vitamin K by the enzyme epoxide reductase. The formation of vitamin K epoxide has sometimes been called an epoxidation reaction. Gla modification is critical for the function of more than a dozen proteins involved in blood coagulation and calcium homeostasis (6, 7). The importance of vitamin K-dependent proteins may be even greater than previously thought, as evidenced by the discovery of growth-arrest protein gas-6 (8), and the very recent identification of four putative vitamin K-dependent membrane Gla proteins PRGP1, PRGP2, TMG3, and TMG4 (9, 10). There are ten cysteine residues in the human carboxylase molecule. Our work on the topology of the carboxylase predicts that of these ten cysteines, two are located in the cytoplasm, three are buried in the ER membrane, and five are found in the lumen of the ER (11). Sulfhydryl groups and disulfide bonds are important for both the structure and function of proteins (12-15). For example, the natural abundance of cysteine is 1.2%, but these residues constitute 5.6% of enzyme catalytic sites (16). Therefore, identification of free cysteine residues or those involved in disulfide bond formation can give valuable information about the structure and function of proteins.Several studies have implicated cysteine in the function of carboxylase. Chemical modification of carboxylase by sulfhydryl-reactive reagents suggests that cysteine residues are important for the carboxylation reaction (17-21). Based on a non-enzymatic chemical model, Paul Dowd et al. (22) developed a "base strength amplification mechanism" for carboxylation. They proposed that two free cysteines are involved in the active site of carboxylase. Recently, Pudota et al. (21) analyzed the catalytically important cysteine residues of the carboxylase by modifying free cysteines with the radiolabeled sulfhydryl-reactive reagent 14 C-NEM. These authors reported that cysteine residues 99 and 450 are the active site residues of carboxylase (21). In contrast to the multiple studies on the importance of free cysteines in carboxylase, the only information about disulfide bridges in the structure of the carboxylase is the study by * This work was supported by National Institutes ...