Identification of Sequences within the γ-Carboxylase That Represent a Novel Contact Site with Vitamin K-dependent Proteins and That Are Required for Activity

Abstract

The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to ␥-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 The vitamin K-dependent (VKD) 1 or ␥-carboxylase converts Glus to ␥-carboxylated Glus (Glas) in VKD proteins as they transit through the endoplasmic reticulum (1, 2). Most of the VKD proteins are secreted out of the cell, and carboxylation of their Gla domain confers the ability to bind phospholipid bilayers, where these proteins exert their effects. Carboxylation is thus required for the biological activity of VKD proteins, which function in hemostasis, calcium homeostasis, and growth control. In addition, a novel subset of mammalian VKD proteins with potential functions in signal transduction has recently been identified by sequence homology (3, 4). Unlike the other VKD proteins, these proteins apparently have a single-pass transmembrane domain with the extracellular domain containing the predicted carboxylated region. Inhibition of VKD protein activities forms the basis of anticoagulant therapies with warfarin and coumadin, in which the carboxylation of hemostatic VKD proteins, as well as the other VKD proteins, is reduced by limiting the supply of vitamin K cofactor to the carboxylase.Although the carboxylase was first identified in mammals, carboxylase homologs and activity have been found in fish, the fish-hunting cone snails of the genus Conus, and the fruit fly Drosophila (5-9). All chordates appear to contain the hemostatic VKD proteins (10). The known VKD proteins of Conus, however, have a distinct function where the VKD proteins are neurotoxic venom peptides (11)(12)(13)(14)(15). VKD proteins have not yet been isolated in Drosophila, and so the function of carboxylation in the fruit fly is not currently known.The carboxylase modifies VKD proteins by using O 2 and vitamin K hydroquinone (KH 2 ) to abstract the ␥-hydrogen of glutamyl residues to form a carbanion intermediate, which then incorporates CO 2 via nucleophilic attack to form the Gla (1, 2). During each Glu to Gla conversion, one molecule of KH 2 is oxidized to vitamin K epoxide, and the carboxylase is also an epoxidase. Insights into the molecular mechanism for this reaction have only recently been revealed. Early studies with thiol-specific inhibitors implicated Cys residues as part of the carboxylase active site (16). Chemical modeling based on those studies led to a proposed base strength amplification mechanism where a weak base (thiolate) initiates KH 2 oxygenation to generate a strong base that can abstract the

Determination of Disulfide Bond Assignment of Human Vitamin K-dependent γ-Glutamyl Carboxylase by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…In contrast to their results, however, we found that C288S had only 56% activity, while they reported that this mutation had no effect on activity. Another discrepancy with their result is that we found that C343S had normal activity, while they found that its activity was significantly reduced compared with wild-type enzyme (35); all the other mutants were fully active.…”

“…Because our results on the activity of carboxylase with point mutations C343S and C288S differ from those of Pudota et al (21,35), we isolated the virus DNA from which these mutant proteins were prepared and the entire cDNA sequence was again confirmed. Carboxylase Activity Assays-The carboxylase activity assay was performed in a total volume of 125 l containing 25 mM MOPS, pH 7.5, 500 mM NaCl, 0.8 M (NH 4 ) 2 SO 4 , 0.12% PC, 0.28% CHAPS, 4 M proFIX, 5 Ci of NaH 14 CO 3 (specific activity, 54 mCi/mmol), 222 M vitamin K hydroquinone, and 1.25 mM FLEEL.…”

Genetic determinants of acenocoumarol and phenprocoumon maintenance dose requirements

“…The Arg325Glu variant is located within a domain of the GGCX gene that mediates enzyme/substrate interactions [45]. Our study also failed to support a substantial role for allelic variants in the APOE gene as determinants of AC/PC maintenance doses, as has been suggested previously.…”

A new model for vitamin K-dependent carboxylation: The catalytic base that deprotonates vitamin K hydroquinone is not Cys but an activated amine

“…Mutation to Ser was chosen because Ser is the most conservative substitution and therefore less likely to destabilize protein structure, which could confound analysis, and because Ser substitution for the Cys catalytic bases can result in large decreases in activity (24)(25)(26)(27)(28). We previously generated several of these mutants [C99S, C139S, C288S, C311S, C343S, and C450S (23,30)], and the remainder were generated in these studies (Supporting Materials and Methods). The specific activities were determined by carboxylase assay and quantitative Western analysis of microsomes.…”

“…Cys-343 lies within a region of the carboxylase that binds VKD proteins (30), and substitution by Ser caused partial loss of activity (Table 3). However, the same specific activity was observed when Cys-343 was substituted by a nonionizable Ala, ruling out the possibility of this Cys serving as the catalytic base.…”