Vitamin K-dependent carboxylase: Influence of the “propeptide” region on enzyme activity

Cheung, Alex; Engelke, Jean A.; Sanders, Cynthia K.; Suttie, J. W.

doi:10.1016/0003-9861(89)90472-4

Cited by 37 publications

(40 citation statements)

References 18 publications

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“…FLEEL Carboxylation-The binding sites for glutamate and propeptide appear to be functionally linked (12,14,22). We therefore investigated the effects of these mutations on the kinetics of CO 2 incorporation into FLEEL.…”

Section: Resultsmentioning

confidence: 99%

“…First they show that in most cases the mutations specifically affect propeptide binding, and second the mutations that do affect catalysis are of interest in terms of linkage between the substrate binding site and the propeptide binding site. Knobloch and Suttie reported that the propeptide of factor X stimulates CO 2 incorporation into FLEEL and suggested that this was due to such a linkage phenomenon (12,14,27). We predicted, based on this hypothesis, that mutation of certain propeptide binding residues will affect the interaction of carboxylase with non-propeptide-containing substrates.…”

Section: Discussionmentioning

confidence: 99%

“…The role of the substrate's propeptide is to anchor it to the carboxylase for a time sufficient for multiple carboxylations (13). In addition to its role in anchoring the substrate to the carboxylase, binding of the propeptide to carboxylase also significantly stimulates the incorporation of CO 2 into non-propeptide-containing substrates (12,14).We have recently demonstrated that, at 20°C, the affinities (measured as K i ) of the various propeptides for carboxylase vary from about 5 nM for factor X to greater than 500 M for bone Gla protein (15). We further demonstrated that a consensus propeptide bound considerably tighter to carboxylase than any known naturally occurring propeptide (16).…”

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The Putative Vitamin K-dependent γ-Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site

Lin

Jin

Tie

et al. 2002

Journal of Biological Chemistry

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The vitamin K-dependent ␥-glutamyl carboxylase binds an 18-amino acid sequence usually attached as a propeptide to its substrates. Price and Williamson (Protein Sci. (1993) 2, 1997-1998) noticed that residues 495-513 of the carboxylase shares similarity with the propeptide. They suggested that this internal propeptide could bind intramolecularly to the propeptide binding site of carboxylase, thereby preventing carboxylation of substrates lacking a propeptide recognition sequence. To test Price's hypothesis, we created nine mutant enzyme species that have single or double mutations within this putative internal propeptide. The apparent K d values of these mutant enzymes for human factor IX propeptide varied from 0.5-to 287-fold when compared with that of wild type enzyme. These results are consistent with the internal propeptide hypothesis but could also be explained by these residues participating in propeptide binding site per se. To distinguish between the two alternative hypotheses, we measured the dissociation rates of propeptides from each of the mutant enzymes. Changes in an internal propeptide should not affect the dissociation rates, but changes to a propeptide binding site may affect the dissociation rate. We found that dissociation rates varied in a manner consistent with the apparent K d values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide and V max . Taken together, our results indicated that these mutations affected the propeptide binding site rather than a competitive inhibitory internal propeptide sequence. These results agree with our previous observations, indicating that residues in this region are involved in propeptide binding.The vitamin K-dependent ␥-glutamyl carboxylase is a polytopic integral membrane protein that resides in the endoplasmic reticulum (1). It catalyzes the post-translational modification of a number of vitamin K-dependent proteins (e.g. the coagulation proteins prothrombin, factor VII, factor IX, factor X, protein S, protein C, and protein Z) (2, 3). Other known vitamin K-dependent proteins are the bone-related proteins osteocalcin and matrix Gla protein, the growth arrest protein Gas 6, and four proteins of unknown function: proline-rich Gla proteins I and II and TMG proteins 3 and 4 (4 -9). Vitamin K-dependent carboxylase utilizes the substrates: reduced vitamin K, carbon dioxide, oxygen, and a propeptide-containing substrate. Multiple glutamic acid residues of the polypeptide substrate, within about 40 residues of the propeptide, are usually modified to ␥-carboxyglutamate during a single binding event (10).The primary interaction between the vitamin K-dependent carboxylase and its substrates is mediated by the 18-amino acid propeptide sequence (11, 12), which in all known vitamin K-dependent proteins, except for matrix Gla protein, is removed prior to secretion. The role of the substrate's propeptide is to anchor it to the carboxylase for a time sufficient for multiple c...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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The Putative Vitamin K-dependent γ-Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site

Lin

Jin

Tie

et al. 2002

Journal of Biological Chemistry

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“…It is significant that the rate of dissociation (3.7 ϫ 10 Ϫ4 s Ϫ1 ) is so similar to the steady state rate constant (4.5 ϫ 10 Ϫ4 s Ϫ1 ) measured above. The Effects of Glutamic Acid-containing Peptides on the Dissociation Rate of ProFIX18 -It is known that binding of the propeptide to the carboxylase reduces the K m of a small substrate (FLEEL) (26,28,29). Moreover, under turnover conditions (in the presence of FLEEL, vitamin K, CO 2 , and O 2 ), both the K d and the k off values of proFIX18 decrease relative to those in the absence of substrates (23).…”

Section: K D Values Of Profix18 Fixprogla41mentioning

confidence: 99%

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

Lin

Straight

Stafford

2004

Journal of Biological Chemistry

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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...

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Biological Function of Vitamin K Antagonists

Suttie

1990

New Trends in Haemostasis

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Vitamin K-dependent carboxylase: Influence of the “propeptide” region on enzyme activity

Cited by 37 publications

References 18 publications

The Putative Vitamin K-dependent γ-Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site

The Putative Vitamin K-dependent γ-Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site

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

Biological Function of Vitamin K Antagonists

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