The Vitamin K-dependent Carboxylase Generates γ-Carboxylated Glutamates by Using CO2 to Facilitate Glutamate Deprotonation in a Concerted Mechanism That Drives Catalysis

Abstract

The ␥-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 ␥-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.

Interaction of 17β-estradiol and dietary fatty acids on energy and glucose homeostasis in female mice

“…Respiratory quotient (CO 2 /O 2 , has also been used as an independent predictor of body mass changes 47 . There is evidence from the basic chemistry of the Bohr effect on hemoglobin 48 , as well as the hypercapnic activation of vitamin K2 dependent enzymes 49 , that CO 2 supports the metabolic rate. Boulder and other high altitude cities 50 have substantially lower rates of obesity and associated metabolic disease.…”

Vitamin K Oxygenation, Glutamate Carboxylation, and Processivity: Defining the Three Critical Facets of Catalysis by the Vitamin K–Dependent Carboxylase

“…Reexamination of the dependence of Glu deprotonation on CO 2 using tritiated peptides (Fig. 8B) and wild-type enzyme revealed that with purified carboxylase, deprotonation has a profound dependence on CO 2 , which closely paralleled the formation of Gla (52). Further, as Glu deprotonation did not occur in the absence of CO 2 , tritium incorporation from tritiated water was barely detected (Fig.…”

The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation

“…This test was possible because VKORC1 is rate-limiting for carboxylation (21,32) and because of a previous development that improved the sensitivity of the carboxylation assay (27). Endogenous levels of VKORC1 in 293 cells are low, and this assay (described below) therefore enabled analysis of VKORC1 activity.…”

“…The reactions were initiated by the addition of KO (65 M), and after a 90-min incubation, the samples were boiled and then subjected to two rounds of chromatography to increase the signal to noise. This approach was required because of the low level of VKORC1 activity in the microsomes, and the details for this method have been described previously (27). Briefly, samples were chromatographed on P-2 columns (Bio-Rad), and carboxylated FLEEL was then isolated by HPLC and quantitated by scintillation counting.…”