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.