Methylation of γ-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the γ-glutamyl carboxylase.

γ-Carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively charged residue. Gla is generated by a single enzyme, the γ-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.

Identification of sequences within the gamma-carboxylase that represent a novel contact site with vitamin K-dependent proteins and that are required for activity.

The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to gamma-carboxylated Glu residues (Glas) in VKD proteins, which is required for their activity. VKD precursors are targeted to the carboxylase by their carboxylase recognition site, which in most cases is a propeptide. We have identified a second tethering site for carboxylase and VKD proteins that is required for carboxylase activity, called the vitamin K-dependent protein site of interaction (VKS). Several VKD proteins specifically bound an immobilized peptide comprising amino acids 343-355 of the human carboxylase (CVYKRSRGKSGQK) but not a scrambled peptide containing the same residues in a different order. Association with the 343-355 peptide was independent of propeptide binding, because the VKD proteins lacked the propeptide and because the 343-355 peptide did not disrupt association of a propeptide factor IX-carboxylase complex. Analysis with peptides that overlapped amino acids 343-355 indicated that the 343-345 CVY residues were necessary but not sufficient for prothrombin binding. Ionic interactions were also suggested because peptide-VKD protein binding could be disrupted by changes in ionic strength or pH. Mutagenesis of Cys(343) to Ser and Tyr(345) to Phe resulted in 7-11-fold decreases in vitamin K epoxidation and peptide (EEL) substrate and carboxylase carboxylation, and kinetic analysis showed 5-6-fold increases in K(m) values for the Glu substrate. These results suggest that Cys(343) and Tyr(345) are near the catalytic center and affect the active site conformation required for correct positioning of the Glu substrate. The 343-355 VKS peptide had a higher affinity for carboxylated prothrombin (K(d) = 5 microm) than uncarboxylated prothrombin (K(d) = 60 microm), and the basic VKS region may also facilitate exiting of the Gla product from the catalytic center by ionic attraction. Tethering of VKD proteins to the carboxylase via the propeptide-binding site and the VKS region has important implications for the mechanism of VKD protein carboxylation, and a model is proposed for how the carboxylase VKS region may be required for efficient and processive VKD protein carboxylation.

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

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.