Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

Abstract

Propeptides of the vitamin K-dependent proteins bind to an exosite on ␥-glutamyl carboxylase; while they are bound, multiple glutamic acids in the ␥-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.The vitamin K-dependent ␥-glutamyl carboxylase is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the post-translational modification of specific glutamic acid residues to ␥-carboxylglutamic acid in a number of vitamin K-dependent proteins. In these vitamin K-dependent proteins, multiple glutamic acid residues in the amino-terminal Gla 1 domain are modified (1-3). The existence of these multiple ␥-carboxylglutamic acid residues allows the Gla domain to form the calcium-dependent conformation required for the activity of these vitamin K-dependent proteins (4, 5). Under-carboxylated vitamin K-dependent proteins cannot form this calcium-dependent structure and, as a result, possess poor affinities for phospholipid surfaces, endothelial cells, or activated platelets (6 -8).Previous studies indicate that all or nearly all of the Glu residues to be carboxylated are modified during a single substrate binding event (9, 10). The loss of as few as three carboxylations can markedly decrease the activities of vitamin K-dependent proteins (8). Therefore, because the most frequently used anticoagulant, warfarin, causes under-carboxylation by reducing vitamin K concentration, understanding the mechanism by which processivity yields functional enzymes is important.A tethered model has been proposed to account for how carboxylase accomplishes full carboxylation of pro-factor IX (9, 11). In this hypothesis, the primary interaction between carboxylase and its substrates is mediated by a propeptide sequence of 18 amino acids. Presumably, the propeptide anchors the Gla domain of the substrate near the carboxylase active site for a time sufficient for modification of all or most of the Glu residues of the Gla domain.Several lines of evidence are consistent with the idea that full carboxylation is the result of a stochastic, processive mechanism, i.e. the degree of carboxylation is controlled by the balance between the carboxylation rate of the substrate and the dissociation rate of the product. First, bone Gla protein, whose propeptide has an extremely low affinity (K i Ͼ 500 M) for the carboxylase and therefore a rapid dis...

Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH2

“…Not only is the turnover number of purified VKOR higher than that measured in microsomes; it is also Ϸ10-fold higher than that of the ␥-glutamyl carboxylase (18). Given that VKOR and carboxylase are integral membrane proteins, and that in vitro assays are far removed from actual in vivo conditions, which at the present are unknown, the VKOR turnover number appears to be adequate for the vitamin K cycle.…”

Effect of Vitamin K-dependent Protein Precursor Propeptide, Vitamin K Hydroquinone, and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

“…As the first binding event it most likely sets the stage for binding of the other substrates. In addition, the turnover rate during carboxylation of peptide substrates depends to a certain extent on the propeptide affinity for the carboxylase (7). For these reasons we felt that the carboxylase-bound propeptide would be a good model for the enzyme vitamin K-dependent protein precursor complex.…”

“…The Effect of Substrates on the Dissociation Rate Constants of Propeptides from Carboxylase-In previous studies from this laboratory, the results showed that under catalytic conditions or with FLEEL, the k off for the propeptide of factor IX was slower than when the propeptide was bound to carboxylase in the absence of substrates (6,7). To determine whether this is a general phenomenon, we determined the k off values for the propeptides of protein C and prothrombin as well as that of factor IX in the presence and absence of FLEEL.…”

“…Presnell et al (6) demonstrated that under catalytic conditions, k off for the propeptide of factor IX was slower than in the absence of catalysis and that the K d for that peptide was lower under catalytic conditions. Lin et al (7) showed that Glu-containing peptides stabilize the propeptide-carboxylase complex. There are other reports that seem to imply that other substrate binding events affect the carboxylase conformation and its activity (4, 8 -10).…”

Unique secretion mode of human protein Z: its Gla domain is responsible for inefficient, vitamin K–dependent and warfarin-sensitive secretion

“…10,11 It is also reported that the Gla domain of FIX contributes allosterically to the interaction between its PP and GCX. 25 The affinities of PPs for GCX vary among Vit.K-dependent proteins by more than 100-fold, and the PP of FX has the highest affinity to GCX of all, at least in vitro. 26 The inhibition of PZ secretion by mini-constructs having the PP of FX may have been due to the more dominant interaction of the FX-PP with the PP-binding site of GCX over PZ, by competing the binding of the PZ-PP to GCX.…”