Abstract
The γ-glutamyl carboxylase converts Glu to carboxylated Glu (Gla) to activate a large number of vitamin K-dependent proteins with diverse functions, and this broad physiological impact makes it critical to understand the mechanism of carboxylation. Gla formation is thought to occur in two independent steps (i.e. Glu deprotonation to form a carbanion that then reacts with CO(2)), based on previous studies showing unresponsiveness of Glu deprotonation to CO(2). However, our recent studies on the kinetic properties of a variant enzyme (H160A) showing impaired Glu deprotonation prompted a reevaluation of this model. Glu deprotonation monitored by tritium release from the glutamyl γ-carbon was dependent upon CO(2), and a proportional increase in both tritium release and Gla formation occurred over a range of CO(2) concentrations. This discrepancy with the earlier studies using microsomes is probably due to the known accessibility of microsomal carboxylase to water, which reprotonates the carbanion. In contrast, tritium incorporation experiments with purified carboxylase showed very little carbanion reprotonation and consequently revealed the dependence of Glu deprotonation on CO(2). Cyanide stimulated Glu deprotonation and carbanion reprotonation to the same extent in wild type enzyme but not in the H160A variant. Glu deprotonation that depends upon CO(2) but that also occurs when water or cyanide are present strongly suggests a concerted mechanism facilitated by His-160 in which an electrophile accepts the negative charge on the developing carbanion. This revised mechanism provides important insight into how the carboxylase catalyzes the reaction by avoiding the formation of a high energy discrete carbanion.
Highlights
DECEMBER 30, 2011 VOLUME 286 NUMBER 52 association with the carboxylase and the processive modification of clusters of Glu residues to carboxylated Glu (Gla) residues in their Gla domains [2, 3]
We found that carboxylase residue His-160 is required for Glu deprotonation [27]; the H160A mutant that revealed the function of His-160 had a puzzling property, which was a 5-fold increase in Km for CO2 and which suggested that Glu deprotonation mediated by His-160 depends upon the presence of CO2
The results show that Glu deprotonation clearly depends upon CO2, and they suggest a concerted mechanism of Gla formation in which Glu deprotonation and CO2 addition occur simultaneously
Summary
Expression and Isolation of Carboxylase—Baculoviruses containing wild type carboxylase or a variant with His-160 substituted by Ala were generated and expressed in SF21 insect cells as previously described [27]. Both enzymes contain a C-terminal addition of an alanine linker followed by the FLAG epitope (i.e. AAADYKDDDDK). After centrifugation (100,000 ϫ g, 4 °C, 1 h), carboxylase was affinity-purified from the solubilized microsomes using agarose-coupled anti-FLAG antibody resin (Sigma). Sample (1 ml) was nutated overnight with anti-FLAG resin (100 l, 0.6 mg/ml), which was washed by five successive rounds of centrifugation (10,000 ϫ g, 1 min) and incubation in 1 ml of 25 mM Tris-HCl, pH 7.4, 0.3% (w/v) CHAPS, 0.2% (w/v) phosphatidylcholine, and 500 mM NaCl, all at 4 °C. Wild type carboxylase was tested in an activity assay and had a specific activity indistinguishable from enzyme isolated from tissue [28], indicating that the recombinant form was fully functional
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