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• W1998117005 abstract "The vitamin K-dependent γ-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 apparentK d values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide andV 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 γ-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 apparentK d values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide andV 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 pentapeptide Phe-Leu-Glu-Glu-Leu 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 3-(N-morpholino)propanesulfonic acid the 19-mer peptide fluorescein-GAVFLSREQANQVLQRRRR modified at its N terminus with 5(6)-carboxyfluorescein the 19-mer peptide fluorescein-TVFLDHENANKILNRPKRY modified at its N terminus with 5(6)-carboxyfluorescein the 19-mer peptide, TVFLDHENANKILNRPKRYFIX(Q/S), 59-amino acid peptide containing human factor IX propeptide, and first 41 residues of FIX Gla domain (sequence −18 to 41) The vitamin K-dependent γ-glutamyl carboxylase is a polytopic integral membrane protein that resides in the endoplasmic reticulum (1Helgeland L. Biochim. Biophys. Acta. 1977; 499: 181-193Crossref PubMed Scopus (34) Google Scholar). 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) (2Suttie J.W. Annu. Rev. Biochem. 1985; 54: 459-477Crossref PubMed Scopus (376) Google Scholar, 3Suttie J.W. FASEB J. 1993; 7: 445-452Crossref PubMed Scopus (108) Google Scholar). 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 (4Manfioletti G. Brancolini C. Avanzi G. Schneider C. Mol. Cell. Biol. 1993; 13: 4976-4985Crossref PubMed Scopus (532) Google Scholar, 5Kulman J.D. Harris J.E. Haldeman B.A. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9058-9062Crossref PubMed Scopus (94) Google Scholar, 6Kulman J.D. Harris J.E. Xie L. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1370-1375Crossref PubMed Scopus (91) Google Scholar, 7Price P.A. Annu. Rev. Nutr. 1988; 8: 565-583Crossref PubMed Scopus (151) Google Scholar, 8Furie B. Bouchard B.A. Furie B.C. Blood. 1999; 93: 1798-1808Crossref PubMed Google Scholar, 9Price P.A. Faus S.A. Williamson M.K. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1400-1407Crossref PubMed Scopus (470) Google Scholar). 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 γ-carboxyglutamate during a single binding event (10Morris D.P. Stevens R.D. Wright D.J. Stafford D.W. J. Biol. Chem. 1995; 270: 30491-30498Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The primary interaction between the vitamin K-dependent carboxylase and its substrates is mediated by the 18-amino acid propeptide sequence (11Pan L.C. Price P.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6109-6113Crossref PubMed Scopus (156) Google Scholar, 12Knobloch J.E. Suttie J.W. J. Biol. Chem. 1987; 262: 15334-15337Abstract Full Text PDF PubMed Google Scholar), 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 carboxylations (13Jorgensen M.J. Cantor A.B. Furie B.C. Brown C.L. Shoemaker C.B. Furie B. Cell. 1987; 48: 185-191Abstract Full Text PDF PubMed Scopus (153) Google Scholar). In addition to its role in anchoring the substrate to the carboxylase, binding of the propeptide to carboxylase also significantly stimulates the incorporation of CO2 into non-propeptide-containing substrates (12Knobloch J.E. Suttie J.W. J. Biol. Chem. 1987; 262: 15334-15337Abstract Full Text PDF PubMed Google Scholar, 14Cheung A. Engelke J.A. Sanders C. Suttie J.W. Arch. Biochem. Biophys. 1989; 274: 574-581Crossref PubMed Scopus (26) Google Scholar). 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 μm for bone Gla protein (15Stanley T.B. Jin D.Y. Lin P.J. Stafford D.W. J. Biol. Chem. 1999; 274: 16940-16944Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We further demonstrated that a consensus propeptide bound considerably tighter to carboxylase than any known naturally occurring propeptide (16Stanley T.B. Humphries J. High K.A. Stafford D.W. Biochemistry. 1999; 38: 15681-15687Crossref PubMed Scopus (24) Google Scholar). Knobloch and Suttie (12Knobloch J.E. Suttie J.W. J. Biol. Chem. 1987; 262: 15334-15337Abstract Full Text PDF PubMed Google Scholar) found that the propeptide of factor X decreased the K m and increased theV max of the carboxylase for the small Glu-containing substrate FLEEL.1 They proposed that this stimulation resulted in propeptide-containing substrates being selectively modified by the carboxylase. Based upon this suggestion, Price and Williamson (17Price P.A. Williamson M.K. Protein Sci. 1993; 2: 1987-1988Crossref PubMed Scopus (15) Google Scholar) searched the carboxylase for a sequence that could compete with the propeptide for its substrates. They searched the 24-amino acid sequence of exon 3 of the human matrix Gla protein and found a sequence within the carboxylase homologous with the propeptide sequence. Based upon the earlier observation that the propeptide stimulated incorporation of CO2 into FLEEL, they postulated that residues 495–513 of the carboxylase functioned as an internal propeptide, and could bind intramolecularly to the propeptide binding site. They further suggested that this intramolecular interaction would prevent carboxylation of proteins lacking the propeptide docking sequence. To test this hypothesis, they proposed that one should reduce the affinity of the internal propeptide for the propeptide binding site by mutating the highly conserved phenylalanine that corresponded to −16 of the propeptide. With our knowledge that the consensus sequence propeptide binds tightly to carboxylase (16Stanley T.B. Humphries J. High K.A. Stafford D.W. Biochemistry. 1999; 38: 15681-15687Crossref PubMed Scopus (24) Google Scholar), we set out to test Price's hypothesis. We hypothesized that if we made the putative internal propeptide sequence more (or less) like the consensus sequence, the intramolecular binding should be tighter (or weaker) and that it would require higher (or lower) concentrations of propeptide to stimulate CO2 incorporation into the small substrate FLEEL. To test the hypothesis that there was an internal propeptide, we made nine single or double mutations in the putative internal propeptide of human carboxylase and tested their effect on the carboxylase molecule. Our results strongly suggest that the proposed internal propeptide contributes to the propeptide binding site rather than to a self-regulatory domain. All chemicals were reagent grade. FLEEL was purchased from Bachem (Philadelphia, PA). CHAPS was from Pierce. Leupeptin, pepstatin, aprotinin, and phenylmethylsulfonyl fluoride were from Roche Molecular Biochemicals. Vitamin K1 was from Sigma and was reduced to hydroquinone (KH2) as previously described (18Morris D.P. Soute B.A. Vermeer C. Stafford D.W. J. Biol. Chem. 1993; 268: 8735-8742Abstract Full Text PDF PubMed Google Scholar). 1,2-Dioleoyl-sn-glycero-3-phosphocholine was from Avanti Polar Lipids (Alabaster, AL). Bovine serum albumin (fraction V, heat shock) was from Roche Molecular Biochemicals. Fluorescein-labeled and unlabeled peptides, based on the human FIX propeptide, proFIX19, and consensus sequences (16Stanley T.B. Humphries J. High K.A. Stafford D.W. Biochemistry. 1999; 38: 15681-15687Crossref PubMed Scopus (24) Google Scholar) were chemically synthesized, purified, and verified by ion spray mass spectrometry by Chiron Mimotopes (Clayton, Victoria, Australia). In the synthesis of the fluorescein-labeled propeptides, 5(6)-carboxyfluorescein was conjugated to the amino terminus of the propeptide. Purity of all peptides was determined by mass spectrometry and high pressure liquid chromatography and was found to be ∼95%. The pSK− vector was from Stratagene (La Jolla, CA). The pVL1392 vector was from Pharmingen (San Diego, CA). The BacVector 3000 baculoviral DNA was from Novagen (Madison, WI). Sf9 insect cells were obtained from the Lineberger Cancer Center at the University of North Carolina (Chapel Hill, NC). High Five insect cells were provided by Dr. Thomas Kost of Glaxo Wellcome. HPC4 antibody affinity resin was obtained from Dr. Charles Esmon (Oklahoma Medical Research Foundation). SP Sepharose was from Amersham Biosciences. All other materials were from Sigma and were reagent grade. The cDNA of human γ-glutamyl carboxylase was cloned into the pSK− vector and was modified to the desired sequence by site-directed mutagenesis as previously described (19Mutucumarana V.P. Stafford D.W. Stanley T.B. Jin D.Y. Solera J. Brenner B. Azerad R. Wu S.M. J. Biol. Chem. 2000; 275: 32572-32577Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). All constructs having carboxylase sequence were flanked by sequences coding for the FLAG (DYKDDDDK) tag at their amino termini and by the HPC4 tag (EDQVDPRLIDGK) (20Stearns D.J. Kurosawa S. Sims P.J. Esmon N.L. Esmon C.T. J. Biol. Chem. 1988; 263: 826-832Abstract Full Text PDF PubMed Google Scholar) at their carboxyl termini. The presence of these tags does not appear to alter the properties of the carboxylase molecule; i.e. its activity and kinetic properties are not measurably different from the wild type enzyme. The DNA sequence of each construct was determined in its entirety by the DNA sequence facility at the University of North Carolina at Chapel Hill. The engineered DNAs were subcloned into the pVL1392 vector, and the proteins were expressed in baculovirus-infected High Five cells as previously described (15Stanley T.B. Jin D.Y. Lin P.J. Stafford D.W. J. Biol. Chem. 1999; 274: 16940-16944Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Solubilization and isolation of microsomes from High Five cells were done as previously described (15Stanley T.B. Jin D.Y. Lin P.J. Stafford D.W. J. Biol. Chem. 1999; 274: 16940-16944Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). All enzyme preparations were purified to homogeneity (purity > 98%) as previously described (15Stanley T.B. Jin D.Y. Lin P.J. Stafford D.W. J. Biol. Chem. 1999; 274: 16940-16944Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The enzyme preparations were preincubated with 0.067 m iodoacetamide at 37 °C for 15 min to eliminate the effects of dithiothreitol on the protein assay reagents. The total protein concentrations (including both active and inactive enzyme) were determined by using Bio-Rad detergent-compatible protein assay kits and IgG protein standard following the supplier's protocol. The active concentration of our enzyme preparations was determined by titration with FproCon as described below. All of the measurements were performed in a 400-μl fluorimetric quartz curette (Starna Cells, Atascadero, CA) at 4.5 °C in a final sample volume of 300 μl. Enzymes and propeptides were preincubated for 1 h in standard buffer containing 100 mm MOPS (pH 7.5), 166 mmNaCl, 3.5% glycerol, 6.3 mm dithiothreitol, 66 μm EDTA, 0.1% 1,2-dioleoyl-sn-glycero-3-phosphocholine, 0.28% CHAPS, and 0.4% bovine serum albumin. The fluorescence intensity of plane polarized light at four mutually perpendicular polarizing filter settings (I VV,I VH, I HV, andI HH) of samples was measured with an SLM-Aminco 8100 spectrofluorimeter equipped with Glan-Thompson polarizing filters in the excitation and emission beams; each point represents the average of six readings. The excitation and emission wavelengths were 490 and 525 nm, respectively. Anisotropy was calculated from the four measured intensities as previously described (21Lakowicz J.R. Principles of Fluoresence Spectroscopy. Plenum, New York1999: 308-309Google Scholar, 22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). The concentration of active enzyme in a purified carboxylase preparation was determined by measuring the number of propeptide binding sites in the preparation. Previous work indicates a correlation between the presence of a propeptide binding site and catalytic activity and indicates that only active enzyme can bind propeptide. Therefore, a measurement of the concentration of propeptide binding sites in a carboxylase preparation is a measurement of the concentration of the active fraction of enzyme (22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). For each sample, the fraction of FproCon bound to carboxylase was determined from the anisotropy value, and the concentration of propeptide binding sites (active enzyme) was determined from the fraction bound versus [carboxylase] plot as previously described (22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). Briefly, when appropriate conditions are chosen (concentration of FproCon ≫ K d), anisotropy of FproCon increases linearly with increasing carboxylase concentration to about 75% saturation. The equivalence point of active carboxylase concentration was determined from the intersection of a linear regression of the data points (up to 75% fraction bound) with a horizontal line determined from the saturation value (100% fraction bound). All enzyme concentrations used in the calculation of kinetic constants were the active enzyme concentrations determined by its ability to bind propeptide (22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). The time course of FproFIX19 release from wild type and mutant carboxylases was measured essentially as described previously (22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). For the wild type or P495V/Q497A, F496A, and Q503R carboxylases, 10 nm active enzyme was preincubated with 25 nm FproFIX19 in standard buffer at 4.5 °C for 1 h to allow the mixture to come to equilibrium. A 350-fold excess of unlabeled proFIX19 was added at time 0. The sample was added to a microcuvette and was placed into the sample holder of the SLM-Aminco 8100 spectrofluorimeter. Intensity measurements were taken at 20-s intervals as previously described (22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar), and the anisotropy at each data point was calculated (21Lakowicz J.R. Principles of Fluoresence Spectroscopy. Plenum, New York1999: 308-309Google Scholar). For the mutants V502A, V502A/P504Q, S510A, and W512A, 100 nm active carboxylase was premixed with 100 nm FproFIX19 to achieve a starting anisotropy value significantly above base line. For the mutants Q503N and P504Q, 50 nm carboxylase was premixed with 100 nmFproFIX19. All off rate experiments were carried out at 4.5 °C, and all data were well fitted to a model of propeptide release described by a single exponential decay, at=(ai−af)exp(−k∗t)+afEquation 1 where a t is the anisotropy at timet, a i and a f are the anisotropies at initial and final time, respectively, k is the first-order rate constant, and t is time. Time course data were fitted to Equation 1 by minimizing the parametersa i, a f, and k. The changes in fluorescent intensity of FproFIX19 upon binding to the wild type and all mutant enzymes were found to be insignificant and did not affect the fraction bound, so rate constants could be accurately determined directly from the anisotropy data (22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). Reactions were performed with 20 nm active carboxylase in a buffer containing 25 mm MOPS (pH 7.5), 500 mm NaCl, 0.16% CHAPS, 0.16% phosphatidylcholine, 222 μm vitamin K hydroquinone, 6 mm dithiothreitol, 10 μCi of NaH14CO3 (specific activity, 54 mCi/mmol), and 1.5 mm FLEEL. The desired concentration of proFIX19 was included in each reaction. Reactions were at 20 °C for 30 min as previously described (19Mutucumarana V.P. Stafford D.W. Stanley T.B. Jin D.Y. Solera J. Brenner B. Azerad R. Wu S.M. J. Biol. Chem. 2000; 275: 32572-32577Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The fraction of bound enzymes (f b) was determined using the following equation: f b = (V obs −V f)/(V sat − Vf). TheV obs, V f, and Vsat refer to FLEEL carboxylation velocity in the presence of a certain concentration of proFIX19, in the absence of proFIX19, and in the presence of saturating proFIX19 respectively. The apparentK d of wild type and mutant carboxylase was determined from f b as previously described (22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar,23Krishnaswamy S. J. Biol. Chem. 1992; 267: 23696-23706Abstract Full Text PDF PubMed Google Scholar). For kinetic studies of FLEEL carboxylation, 20 nm active carboxylase was used for each reaction. Reactions were performed in the presence or absence of saturating proFIX19 at 20 °C for 30 min and were carried out as described above, except that the concentration of the substrate FLEEL was varied from 0 to 40 mm for V502A and S510A and from 0 to 20 mm for other mutants in the absence of proFIX19. In the presence of saturating proFIX19, the concentration of the substrate FLEEL was varied from 0 to 4.8 mm. For kinetic studies of propeptide-containing substrates, FIX Q/S, consisting of residues −18 to +41 of human FIX with mutations at −4 (Arg → Gln), −1 (Arg → Ser), and 19 (Met → Ile), was used. Previous studies have shown that there is no significant difference in kinetic properties between a wild type substrate and the FIX Q/S (24Wu S.M. Soute B.A. Vermeer C. Stafford D.W. J. Biol. Chem. 1990; 265: 13124-13129Abstract Full Text PDF PubMed Google Scholar). Twenty nm active carboxylase was used for each reaction. Reactions were performed at 20 °C for 60 min in a buffer containing 25 mm MOPS (pH 7.5), 500 mm NaCl, 0.16% CHAPS, 0.16% phosphatidycholine, 222 μm vitamin K hydroquinone, 6 mm dithiothreitol, and 10 μCi of NaH14CO3. The concentration of the propeptide-containing substrate FIX Q/S, was varied from 0 to 2500 nm. To measure the rate of FproCon to carboxylase, 150 nm of wild type, V502A, S510A, or W512A enzyme was added to standard buffer containing 10 nmFproCon at time 0. 20 s after adding enzymes, the anisotropy values were measured at 5-s intervals. The anisotropy at each time point was converted to fraction bound of FproCon as previously described (21Lakowicz J.R. Principles of Fluoresence Spectroscopy. Plenum, New York1999: 308-309Google Scholar, 22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). Our previous results indicate that once FproCon bound to wild type, V502A, S510A, or W512A carboxylase, it would not come off within 12 h (data not shown). Thus, the contributions to the changes of anisotropy in our experiments of the release of FproCon from carboxylase can be ignored. All measurements were performed at 4.5 °C, and all data were fitted to a pseudo-first order reaction equation, c=b∗(1−exp(−k∗a∗t))Equation 2 where c represents the concentration of carboxylase-FproCon complex, b is the total concentration of FproCon, a is the total concentration of enzyme,t is time in seconds, and k is the rate constant. To investigate the function of the putative internal propeptide region of carboxylase, we made several mutations in the region identified by Price and Williamson. These mutations were designed to make the putative internal propeptide sequence either more like or less like a consensus propeptide sequence. The consensus sequence, which was determined by selecting the most prevalent amino acid at each position in all known propeptides, binds to the carboxylase with an affinity much higher than any known, naturally occurring, propeptide (16Stanley T.B. Humphries J. High K.A. Stafford D.W. Biochemistry. 1999; 38: 15681-15687Crossref PubMed Scopus (24) Google Scholar). Fig. 1demonstrates the alignment of the proposed internal propeptide region with the consensus human propeptide and the corresponding region of the mutations used in this paper. If region 495–513 contains an internal propeptide, then mutations rendering it more like the consensus sequence (e.g. V502A, Q503N, P504Q, and the double mutations P495V/Q497L and V502A/P504Q) would be expected to cause stronger intramolecular interactions with the propeptide binding site. These mutations would cause a decrease in the apparent affinity for the propeptide. Conversely, mutations making the region diverge from the consensus sequence (e.g. F496A and Q503R) should reduce the binding between the propeptide binding site and the putative internal propeptide, resulting in an apparent increased affinity for propeptide. S510A and W512A were mutated to test the alternative hypothesis. The free propeptide of the vitamin K-dependent proteins has been shown to stimulate the rate of carboxylation of FLEEL (12Knobloch J.E. Suttie J.W. J. Biol. Chem. 1987; 262: 15334-15337Abstract Full Text PDF PubMed Google Scholar); therefore, the apparentK d of propeptide can be determined by measuring the velocities of FLEEL carboxylation at varied propeptide concentrations. Fig. 2 shows a plot of enzyme fraction bound versus proFIX19 concentrations. The apparentK d of proFIX19 for wild type enzyme was 7.3 ± 0.5 nm, whereas the apparent K d for mutant enzymes varied from 4.2 ± 0.1 to 2200 ± 50 nm (Table I). However, P495V/Q497A and F496A were not significantly different from wild type enzyme (Table I). It is notable that the maximum stimulated rates of FLEEL carboxylation were comparable for all mutants and wild type enzyme.Table IThe apparent Kd for proFIX19 and the maximal stimulated FLEEL carboxylation velocities for wild type and mutant carboxylaseEnzymeApparentK d for proFIX19DifferenceMaximal stimulated activityDifferencenm-foldpmol14CO2 /min/pmol enzyme-foldWild type7.3 ± 0.51.029.1 ± 1.11.0P495V/Q497A9.4 ± 1.31.326.2 ± 2.60.9F496A8.8 ± 0.71.227.1 ± 1.60.9V502A53.7 ± 3.17.428.9 ± 1.31.0V502A/P504Q51.2 ± 4.97.028.2 ± 1.21.0Q503N24.4 ± 1.73.328.0 ± 1.21.0Q503R4.2 ± 0.10.626.6 ± 0.40.9P504Q11.7 ± 1.51.628.0 ± 1.61.0S510A2200 ± 5028725.9 ± 0.90.9W512A1770 ± 5824228.9 ± 1.21.0 Open table in a new tab The binding sites for glutamate and propeptide appear to be functionally linked (12Knobloch J.E. Suttie J.W. J. Biol. Chem. 1987; 262: 15334-15337Abstract Full Text PDF PubMed Google Scholar, 14Cheung A. Engelke J.A. Sanders C. Suttie J.W. Arch. Biochem. Biophys. 1989; 274: 574-581Crossref PubMed Scopus (26) Google Scholar, 22Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). We therefore investigated the effects of these mutations on the kinetics of CO2 incorporation into FLEEL. Kinetic constants for FLEEL carboxylation were determined in the presence or absence of saturating proFIX19. In the absence of proFIX19, Q503R (which has the highest affinity for proFIX19) was found to have the lowestK m (1.90 mm). In contrast, theK m values for FLEEL of V502A, Q503N, and S510A vary from 4.7 to more than 30 mm and are higher than the 3.33 mm of wild type enzyme. The affinity of these mutants (V502A, Q503N, Q503R, and S510A) for FLEEL appears to increase or decrease in parallel with their affinities for proFIX19 (Fig.3 a and TableII). As shown in Fig. 3, although the affinities in the absence of propeptide (inferred from theK m values toward FLEEL) are different from that of wild type enzyme, their V max values are similar to that of the wild type enzyme in the presence of saturating proFIX19. The kinetic constants for FLEEL carboxylation in the presence of saturating proFIX19 were also determined. Fig. 3 bdemonstrates that, in the presence of saturating proFIX19, theK m and V max values of all mutants were similar to wild type enzyme. It is notable that, except for S510A and W512A, the V max values of most mutations and the wild type enzyme were consistently increased 3-fold when the mutant enzymes were saturated with proFIX19 (Table II).Table IIComparison of kinetic parameters for FLEELEnzymeFLEEL without proFIX19FLEEL with saturating proFIX19K mV maxK mV maxmmpmol14CO2 /min/pmol enzymemmpmol14CO2 /min/pmol enzymeWild type3.3 ± 0.417.0 ± 2.60.78 ± 0.1250.4 ± 1.7P495V/Q497A3.8 ± 0.316.3 ± 2.00.82 ± 0.0748.0 ± 2.6F496A3.6 ± 0.217.5 ± 3.00.84 ± 0.0948.2 ± 2.0V502A8.0 ± 0.116.5 ± 0.80.79 ± 0.0749.6 ± 1.2V502A/P504Q7.6 ± 0.716.5 ± 2.10.80 ± 0.1648.7 ± 2.9Q503N4.7 ± 0.415.6 ± 1.50.79 ± 0.0446.0 ± 2.6Q503R1.9 ± 0.217.2 ± 2.50.78 ± 0.1151.8 ± 3.7P504Q3.4 ± 0.316.5 ± 3.50.79 ± 0.1348.6 ± 3.3S510A>308.1 ± 0.80.93 ± 0.0847.4 ± 1.1W512A3.2 ± 0.22.0 ± 0.20.76 ± 0.0638.2 ± 2.8 Open table in a new tab Thus far, our data are consistent with Price's hypothesis (17Price P.A. Williamson M.K. Protein Sci. 1993; 2: 1987-1988Crossref PubMed Scopus (15) Google Scholar). However, we might obtain the same effects if we were mutating the propeptide binding site instead of an internal propeptide. We realized that if mutations were in the proposed internal propeptide site, then, neglecting linkage, they should not affect the off rate of a bound propeptide. If, however, the region mutated was part of the propeptide binding site, the off rate might be affected. As shown in Fig.4, the time course of anisotropy loss due to FproFIX19 release can be satisfactorily fit to a single exponential decay curve, which allows us to determine the off rate constants. The off rate constant for FproFIX19 release from wild type carboxylase was found to be 6.77 ± 0.19 × 10−4s−1, whereas the rate constants for mutants varied from 3.46 ± 0.12 × 10−4 s−1 for Q503R" @default.
• W1998117005 created "2016-06-24" @default.
• W1998117005 creator A5045672145 @default.
• W1998117005 creator A5067697126 @default.
• W1998117005 creator A5072765407 @default.
• W1998117005 creator A5083380392 @default.
• W1998117005 creator A5087713399 @default.
• W1998117005 creator A5088326447 @default.
• W1998117005 date "2002-08-01" @default.
• W1998117005 modified "2023-10-17" @default.
• W1998117005 title "The Putative Vitamin K-dependent γ-Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site" @default.
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