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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 (KH2 ) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO2 , generating the γ-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. &amp; Naganathan, S. (1995)Science 269, 1684–1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH2 ) 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 KH2 preincubation. Amino acid analysis of14 C-N- ethyl maleimide-modified human carboxylase revealed 1.8–2.3 reactive residues and a specific activity of 7 × 108 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 theK 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 KH2 oxygenation.

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