Brønsted Analysis Reveals Lys218 as the Carboxylase Active Site Base That Deprotonates Vitamin K Hydroquinone To Initiate Vitamin K-Dependent Protein Carboxylation

Abstract

The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Brønsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.

Insight into the Coupling Mechanism of the Vitamin K-Dependent Carboxylase: Mutation of Histidine 160 Disrupts Glutamic Acid Carbanion Formation and Efficient Coupling of Vitamin K Epoxidation to Glutamic Acid Carboxylation

“…His160 is one of three His residues conserved between metazoan and Leptospira VKD carboxylases (20); however, mutants with Ala substitutions for the remaining two residues (i.e. H287A and H381A) showed wildtype levels in both epoxidation (26) and carboxylation (data not shown). The results for H160A therefore implicated His160 as important in the coupling of epoxidation to carboxylation.…”

“…All assays were performed in duplicate and included a negative control, which was a sample obtained by performing the anti-FLAG affinity purification on mock-infected insect cells that lack carboxylase. To determine the specific activity of epoxidation, the amount of carboxylase protein was measured in a quantitative Western using an antibody-linked fluorescence assay, as previously described (26). …”

“…Lys218 in the carboxylase deprotonates KH 2 to form KH − (26), which reacts with O 2 to generate a high-energy vitamin K base (K − ). K − then deprotonates Glu to produce a carbanion intermediate that reacts with CO 2 to form Gla, while K − collapses to the KO product.…”

“…An important advance in defining the mechanism has been the identification of Lys218 as the catalytic base that initiates the reaction by deprotonating KH 2 to allow reaction with O 2 and formation of the high-energy vitamin K intermediate (Fig. 1)(26). …”

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

“…How the carboxylase facilitates catalysis is largely unknown because it is a large, integral membrane protein with-out structural information that reveals functional residues (9). A critical breakthrough in understanding the mechanism and in beginning to define the active site was the identification of Lys-218 as the weak base that initiates the reaction by deprotonating KH 2 to generate the vitamin K base (25).…”