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 gamma-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- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr 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(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine 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.

Figure 1

Purification and ¹⁴C-NEM modification of carboxylase. Carboxylase purified in the absence of reducing agents was assayed for enzyme activity (supplemental data, Methods) and modified with ¹⁴C-NEM (Materials and Methods). Duplicate aliquots were loaded on the PhosphorImager gel for quantitation of radioactivity. ¹⁴C-BSA standards were processed in parallel (not shown) to quantitate the amount of ¹⁴C-NEM incorporation (907 cpm in the band) into a known amount of carboxylase activity (1.5 × 10^-4 μmol/min). The preparation shown here also was analyzed by amino acid analysis.

Figure 2

Identification of ¹⁴C-NEM-labeled tryptic peptides of the human carboxylase. A tryptic digest of ¹⁴C-NEM-modified carboxylase was subjected to LC-ESMS, and postcolumn fractions were split with half of the sample being analyzed by mass spectrometry and aliquots (12% of the total digest) of the remainder analyzed by scintillation counting. The ¹⁴C-NEM-labeled peptides identified by mass spectrometry in the radioactive peaks are indicated and described in Table 2.

Figure 3

Functional mapping of the carboxylase. A linear representation of the human carboxylase is shown, along with the two active site Cys residues identified in these studies, and regions that cross link to FLEELY or propeptide, identified in previous biochemical mapping studies (–11). A hydropathy plot also is shown. Analysis was performed at bioinformatics.weizmann.ac.il/hydroph/plot_hydroph.html, using the Kyte-Doolittle x-1 method of calculating hydrophilicty over a window length of 25 to predict membrane spanning regions. The black shading highlights very hydrophobic regions of the carboxylase.