Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation

Abstract

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH 2) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO 2, generating the ␥-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684 -1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH 2) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH 2 preincubation. Amino acid analysis of 14 C-Nethyl maleimide-modified human carboxylase revealed 1.8 -2.3 reactive residues and a specific activity of 7 ؋ 10 8 cpm͞hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0.2% (Cys-99) or 1% (Cys-450), and increased the K ms for a glutamyl substrate 6-to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine͞serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH 2 oxygenation. T he vitamin K-dependent (VKD) carboxylase is an integral membrane enzyme required for the biological activity of proteins involved in hemostasis (prothrombin, factor X, factor VII, factor IX, protein S, protein C, protein Z), calcium homeostasis (bone gla protein and matrix gla protein), cell growth control (gas 6), and possibly signal transduction (PRGP-1 and PRGP-2) (1, 2). Carboxylation is effected via a homologous Ϸ18-aa sequence in VKD proteins, usually an N-terminal propeptide, which the carboxylase binds with high affinity. Propeptide binding of VKD proteins to the carboxylase leads to the conversion of clusters of glutamyl (Glu) residues to ␥-carboxylated glutamyl (or Gla) residues, in a region adjacent to the propeptide called the Gla domain. This domain serves as a calcium-dependent membrane-binding module for the attached VKD proteins, for example to effect blood coagulation on cell surfaces. Carboxylation requires a continual supply of the reduced form of the vitamin K cofactor, vitamin K hydroquinone (KH 2 ), and when KH 2 is limiting undercarboxylated, inactive VKD proteins are produced. Consequently, understanding the mechanism of carboxylation has important medical ramifications as...

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

“…Three of these five mutant enzymes, W390A, S398A, and H404A, exhibited normal activity. To date, only certain cysteine residues have been identified as part of the carboxylase active site (35,36). In addition to cysteines, the participation of other amino acids in catalysis by a multisubstrate enzyme, such as carboxylase, seems likely.…”

r-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

“…4), and a quantitative western showed that the amount of uncarboxylated fIX represented 65% of the intracellular population and corresponded to 7.04 pmol/mg lysate. The intracellular carboxylase concentration could also be quantitated because the specific activity of the carboxylase is known (determined by comparing carboxylase activity of pure enzyme to the amount of protein quantitated by amino acid analysis, (23)). An activity assay on the lysates indicated a value of 0.16 pmol/mg, showing that the intracellular stoichiometry of the carboxylase to uncarboxylated fIX was 1:44.…”

“…Table 1 Levels of fIX, carboxylase and VKOR in fIX BHK and VKOR·fIX BHK cells Lysates were prepared from BHK cells, cultured in the absence of vitamin K, that express low levels of fIX (fIX BHK) or that also express r-VKORC1 (VKOR·fIX BHK). FIX levels were measured in a quantitative western and carboxylase levels were determined by an activity assay and reference to the known specific activity (23). VKOR levels were quantitated by an activity assay that measures KO reduction.…”

“…Most (75%) of the organic phase was transferred to a new tube and the sample was taken to dryness under nitrogen. Samples were resuspended in ethanol and vitamin K was analyzed by HPLC as before (23), along with vitamin K standards (250 ng), to determine the per cent conversion of vitamin K epoxide to quinone. A control reaction with heat-treated extracts showed that KO reduction was not simply due to DTT.…”

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

“…Chemical modification of carboxylase by sulfhydryl-reactive reagents suggests that cysteine residues are important for the carboxylation reaction (17)(18)(19)(20)(21). Based on a non-enzymatic chemical model, Paul Dowd et al (22) developed a "base strength amplification mechanism" for carboxylation.…”