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

Abstract

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...

Vitamin K-dependent proteins in Ciona intestinalis , a basal chordate lacking a blood coagulation cascade

“…DQ900938) with GGC sequences from a variety of vertebrate and invertebrate species (Fig. 8, which is published as supporting information on the PNAS web site) showed a high degree of conservation within, although not restricted to, the five established membrane-spanning regions (31), a previously identified GGC ''fingerprint'' region (32), and an internal propeptide-like sequence that is implicated in recognition of the substrate propeptide (33). Pairwise sequence comparisons (Fig.…”

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

“…Dissociation Rate Measurement-We measured the time course of fluorescein-labeled peptide release from carboxylase as described previously (23,26). We pre-incubated 20 nM fluorescein-labeled peptide with 100 nM carboxylase in buffer A at 17°C for 1 h to allow the mixture to come to equilibrium and then added a 200-fold excess of unlabeled peptide (4 M) at time 0.…”

“…This burst size represents the concentration of 14 CO 2 incorporated into 25 nM FIXproGla41 in one round of reaction (10). The kinetic studies of FLEEL, EEL, and t-butoxycarbonyl-Glu-t-butyl ester carboxylation were performed in buffer A using 25 nM active carboxylase at 17°C for 30 min as described previously (23,26).…”

“…The fraction of bound fluorescein-labeled peptide and the K d values were determined from anisotropy, as described previously (23,26).…”

“…The excitation and emission wavelengths were 490 and 525 nm, respectively. Anisotropy was measured with an OLIS modified T-format SLM spectrofluorimeter (On-Line Instruments, Bogart, GA) essentially as described (23,26).…”