Chemical Modification of Cysteine Residues Is a Misleading Indicator of Their Status as Active Site Residues in the Vitamin K-dependent γ-Glutamyl Carboxylation Reaction
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m408945200
ISSN1083-351X
AutoresJian‐Ke Tie, Da-Yun Jin, David R. Loiselle, R. Marshall Pope, David L. Straight, Darrel W. Stafford,
Tópico(s)Vitamin C and Antioxidants Research
ResumoThe enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to γ-carboxy-glutamic acid by the integral membrane enzyme, γ-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys323 and Cys343. According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys323 and Cys343 may disrupt the three-dimensional structure, resulting in inactivation. The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to γ-carboxy-glutamic acid by the integral membrane enzyme, γ-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys323 and Cys343. According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys323 and Cys343 may disrupt the three-dimensional structure, resulting in inactivation. Vitamin K-dependent carboxylation is required for the biological activity of vitamin K-dependent proteins. Carboxylation is a post-translational conversion of glutamyl residues in the precursor proteins to γ-carboxyglutamic acid (gla) 1The abbreviations used are: gla, γ-carboxyglutamic acid; VKH2, vitamin K hydroquinone; DTT, dithiothreitol; NEM, N-ethylmaleimide; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt; DABMI, 4-dimethylaminophenylazophenyl-4′-maleimide; CHAPS, (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; MOPS, 3-(N-morpholino)propanesulfonic acid; FLEEL, the peptide Phe-Leu-Glu-Glu-Leu; proFIX, 19 amino acid residues of factor IX propeptide (–18 to 1); proFIXgla, propeptide with the gla domain of factor IX (–18 to 46 with the mutations of R-4Q and R-1S); proCon, consensus propeptide (AVFLSREQANQVLQRRRR); MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry. residues in the mature protein (1Vermeer C. Biochem. J. 1990; 266: 625-636Crossref PubMed Scopus (283) Google Scholar, 2Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (991) Google Scholar). These gla residues allow the vitamin K-dependent proteins to bind calcium necessary for their biological functions in blood coagulation, bone homeostasis, and other areas. Vitamin K-dependent carboxylation is catalyzed by an integral membrane glycoprotein, γ-glutamyl carboxylase. During the process of carboxylation, the γ-hydrogen of the glutamic acid is abstracted, followed by the addition of CO2 (1Vermeer C. Biochem. J. 1990; 266: 625-636Crossref PubMed Scopus (283) Google Scholar). Simultaneously, carboxylase converts vitamin K hydroquinone (VKH2) to vitamin K 2,3-epoxide (vitamin K epoxidation) (3Morris D.P. Soute B.A. Vermeer C. Stafford D.W. J. Biol. Chem. 1993; 268: 8735-8742Abstract Full Text PDF PubMed Google Scholar, 4Wallin R. Suttie J.W. Arch. Biochem. Biophys. 1982; 214: 155-163Crossref PubMed Scopus (14) Google Scholar). Numerous publications have suggested that free cysteine residues are important for vitamin K-dependent carboxylation and epoxidation. For example, the addition of DTT to carboxylase-containing microsomes stimulates carboxylation of glutamyl residues (5Friedman P.A. Shia M. Biochem. Biophys. Res. Commun. 1976; 70: 647-654Crossref PubMed Scopus (75) Google Scholar, 6Mack D.O. Suen E.T. Girardot J.M. Miller J.A. Delaney R. Johnson B.C. J. Biol. Chem. 1976; 251: 3269-3276Abstract Full Text PDF PubMed Google Scholar, 7Suttie J.W. Lehrman S.R. Geweke L.O. Hageman J.M. Rich D.H. Biochem. Biophys. Res. Commun. 1979; 86: 500-507Crossref PubMed Scopus (61) Google Scholar, 8Canfield L.M. Sinsky T.A. Suttie J.W. Arch. Biochem. Biophys. 1980; 202: 515-524Crossref PubMed Scopus (25) Google Scholar). Moreover, modification of carboxylase by sulfhydryl-reactive reagents inactivates the enzyme (3Morris D.P. Soute B.A. Vermeer C. Stafford D.W. J. Biol. Chem. 1993; 268: 8735-8742Abstract Full Text PDF PubMed Google Scholar, 8Canfield L.M. Sinsky T.A. Suttie J.W. Arch. Biochem. Biophys. 1980; 202: 515-524Crossref PubMed Scopus (25) Google Scholar, 9Canfield L.M. Biochem. Biophys. Res. Commun. 1987; 148: 184-191Crossref PubMed Scopus (19) Google Scholar, 10Bouchard B.A. Furie B. Furie B.C. Biochemistry. 1999; 38: 9517-9523Crossref PubMed Scopus (19) Google Scholar, 11Pudota B.N. Miyagi M. Hallgren K.W. West K.A. Crabb J.W. Misono K.S. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13033-13038Crossref PubMed Scopus (35) Google Scholar). Canfield (9Canfield L.M. Biochem. Biophys. Res. Commun. 1987; 148: 184-191Crossref PubMed Scopus (19) Google Scholar) reported that a thiol group is essential for the binding of VKH2 to the active site of carboxylase. Bouchard et al. (10Bouchard B.A. Furie B. Furie B.C. Biochemistry. 1999; 38: 9517-9523Crossref PubMed Scopus (19) Google Scholar) showed that NEM, a sulfhydryl-reactive reagent, reacts with two to three free cysteines in bovine carboxylase, resulting in the inactivation of the enzyme. Further support for this hypothesis was provided by Pudota et al. (11Pudota B.N. Miyagi M. Hallgren K.W. West K.A. Crabb J.W. Misono K.S. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13033-13038Crossref PubMed Scopus (35) Google Scholar), who used electrospray mass spectrometry to identify the cysteine residues modified by NEM. They concluded that Cys99 and Cys450 are the two active site cysteines required for both vitamin K-dependent carboxylation and epoxidation. Based on the observations that carboxylase is inactivated by sulfhydryl-reactive reagents, Dowd et al. (12Dowd P. Hershline R. Ham S.W. Naganathan S. Science. 1995; 269: 1684-1691Crossref PubMed Scopus (112) Google Scholar) used a nonenzymatic chemical model to develop a "base strength amplification mechanism" for vitamin K-dependent carboxylation. Those investigators proposed that two free cysteines are involved in the active site of the carboxylase. We recently found that Cys99 and Cys450 are actually joined in a disulfide bond (13Tie J.K. Mutucumarana V.P. Straight D.L. Carrick K.L. Pope R.M. Stafford D.W. J. Biol. Chem. 2003; 278: 45468-45475Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition, we found that mutation of each of the other eight cysteine residues in carboxylase did not inactivate the enzyme, although C288S had only 56% activity relative to that of wild type. Although we confirmed that NEM modification of carboxylase causes inactivation, mutation of the modified residues Cys139, Cys311, Cys323, and Cys343 did not inactivate the carboxylase (13Tie J.K. Mutucumarana V.P. Straight D.L. Carrick K.L. Pope R.M. Stafford D.W. J. Biol. Chem. 2003; 278: 45468-45475Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). These results raise the question of whether sulfhydryl groups are really important for catalysis, and if so by what mechanism? In our first study we changed cysteine to serine in most cases, so here we made cysteine to alanine changes. In addition to mutations, we employed thiol-reactive reagents with different charge/polarity characteristics to identify which cysteine residues in the carboxylase affect the enzyme activity by chemical modification. We used MALDI-TOF MS to identify the chemically modified cysteine residues. Our results suggest that modification of cysteines probably causes structural or steric effects on carboxylase rather than modifying residues directly involved in the carboxylation reaction. While this paper was under review, Rishavy et al. (14Rishavy M.A. Pudota B.N. Hallgren K.W. Qian W. Yakubenko A.V. Song J.H. Runge K.W. Berkner K.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13732-13737Crossref PubMed Scopus (25) Google Scholar) reached a similar conclusion: that cysteine is not an active site residue of vitamin K-dependent carboxylase. Materials—All of the chemicals were reagent grade. AMS and DABMI were obtained from Molecular Probes (Eugene, OR). NEM, phenylmethylsulfonyl fluoride, α-cyano-4-hydroxysuccinnamic acid, Na2S4O6, NaBH4, and CHAPS were obtained from Sigma. Pentapeptide FLEEL and protease inhibitors, H-d-Phe-Phe-Arg-chloromethylketone and H-d-Phe-Pro-Arg chloromethylketone were from Bachem (King of Prussia, PA). 1,2-Dioleoyl-sn-glycero-3-phosphocholine was from Avanti (Alabaster, AL). The propeptide of proFIX and proCon and the fluorescein-labeled proFIX and proCon were chemically synthesized and purified by Chiron Mimotopes (Clayton, Australia). proFIXgla was expressed and purified from Escherichia coli as described previously (15Wu S.M. Soute B.A. Vermeer C. Stafford D.W. J. Biol. Chem. 1990; 265: 13124-13129Abstract Full Text PDF PubMed Google Scholar). Vitamin K1 (10 mg/ml, which contains 70 mg/ml polyxyethylated fatty acid derivative, 37.5 mg/ml dextrose hydrous, and 9 mg/ml benzyl alcohol) was from Abbott Laboratories (Chicago, IL). Aprotinin, pepstatin A, trypsin, and proteinase K were purchased from Roche Applied Science. Polyvinylidene fluoride transfer membrane was from Millipore Co. (Bedford, MA). NaH14CO3 (specific activity, 54 mCi/mmol) was from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Restriction enzymes and peptide N-glycosidase F were from New England Biolab (Beverly, MA). Anti-HPC4 resin was kindly provided by Dr. Charles Esmon (Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK). Reduction of Vitamin K1 to VKH2—Three volumes of buffer K (500 mm NaCl, 20 mm Tris-HCl, pH 8.5) containing 200 mm DTT were added to one volume of vitamin K1 solution (10 mg/ml) and incubated in the dark at 37 °C for at least 24 h to ensure complete reduction. The concentration of this reduced VKH2 stock solution was 5.55 mm. For experiments where DTT was avoided, vitamin K1 was freshly reduced by NaBH4 as follows: vitamin K1 was diluted with an equal volume of buffer K, and NaBH4 was added to the solution to obtain a final concentration of 0.1 m. The reduction reaction was incubated in the dark at room temperature for 20 min. Excess NaBH4 was removed by adjusting the reaction to pH 4.0 with 1 m HCl and incubating at room temperature for 5 min. Then the pH of the solution was adjusted to pH 8.5 with 1 m Tris, and buffer K was added to adjust the concentration of VKH2 to 5.55 mm. Carboxylase Activity Assays—The carboxylase activity was determined by the incorporation of 14CO2 into the pentapeptide substrate FLEEL. The assay was performed in 25 mm MOPS (pH 6.8), 500 mm NaCl, 0.8 m (NH4)2SO4, 0.12% 1,2-dioleoyl-sn-glycero-3-phosphocholine, 0.28% CHAPS (buffer A), with 4 μm proFIX, and 1.25 mm FLEEL. The reaction was started by the addition of 10 μl of an ice-cold mix of NaH14CO3 (final concentration, 40 μCi/ml) and VKH2 (final concentration, 222 μm) to bring the volume to 125 μl. The reaction mix was immediately transferred to a 20 °C water bath and incubated for 30 min. The reactions were terminated by the addition of 1 ml of 5% trichloroacetic acid, and the amount of 14CO2 incorporation was determined as previously described (16Stanley 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). Sulfhydryl-reactive Reagent Modification of Carboxylase—Purified carboxylase (50 nm) (16Stanley 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) was modified by the sulfhydryl-reactive reagents in buffer A. The reaction was initiated by the addition of the sulfhydryl-reactive reagent, and samples were incubated on ice for 20 min in the dark. The reaction was stopped by the addition of DTT to a final concentration of 50 mm. Carboxylase activity was determined by the incorporation of 14CO2 into the pentapeptide substrate FLEEL (16Stanley 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). Na2S4O6 modification is reversed by DTT. Therefore, when we used Na2S4O6 as the thiol-reactive reagent, we stopped the reaction by diluting the sample 10-fold with buffer A. The enzyme activity was determined immediately as described above except we used NaBH4-reduced VKH2. For sequential modification of carboxylase by Na2S4O6 and AMS, carboxylase was first incubated with Na2S4O6 (5 mm, on ice for 10 min). A portion of the sample was incubated with 1 mm AMS for another 10 min. When we wanted to reverse the Na2S4O6 modification, we stopped the reaction by the addition of DTT (final concentration, 50 mm); otherwise we stopped the reaction with dilution as described above. The residual carboxylase activity was measured as above. Limited Trypsinization of Carboxylase—Limited trypsin digestion of carboxylase was performed as previously described (17Wu S.M. Mutucumarana V.P. Geromanos S. Stafford D.W. J. Biol. Chem. 1997; 272: 11718-11722Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A pilot reaction was run for each batch of trypsin and carboxylase to determine the optimal conditions for limited cleavage. Cleavage was accomplished on ice in the presence of 5 μm proFIX. We omitted proFIX in the trypsin digestion samples used for propeptide binding and protection experiments. Trypsin cleavage was stopped by the addition of H-d-Phe-Pro-Arg chloromethylketone and aprotinin to a final concentration of 1.6 μm. We analyzed the extent of trypsin cleavage of carboxylase on gradient (4–20%, reducing conditions) SDS-PAGE gels followed by silver staining. This limited trypsin-digested carboxylase is still enzymatically active. Protection of Carboxylase from Inactivation by Na2S4O6—We investigated whether the following molecules would protect carboxylase from inactivation by Na2S4O6: 10 mm FLEEL, 8 μm proFIX, 8 μm proFIXgla, 222 μm VKH2, and various combinations of these reagents. Carboxylase (50 nm) was incubated with the reagent(s) on ice for 5 min in buffer A. The samples were divided in equal volumes, and Na2S4O6 (500 μm) was added to one half, and solvent/buffer was added to the other. The mixtures were incubated on ice for 20 min, and then carboxylase activity was determined using NaBH4-reduced VKH2. We measured the time course of inactivation of carboxylase and trypsin-digested carboxylase (50 nm) in the presence (8 μm proFIX) or absence of propeptide. We incubated the enzyme with or without propeptide on ice for 5 min. Na2S4O6 was added, portions were removed, and the reaction was stopped at various times. The residual activity was determined as described above. Fluorescence Titration of Intact and Trypsin-digested Carboxylase— Fluorescence anisotropy titration of intact and trypsin-digested carboxylase with 5(6)-carboxyfluorescein-labeled proFIX were performed on an OLIS-modified T-format SLM spectrofluorimeter (On-Line Instruments, Bogart, GA) essentially as described previously (18Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). The excitation wavelength was set as 495 nm, and the slits were set as 4-nm band pass. Two photon counters were used to detect the emissions at horizontal and vertical directions to increase the sensitivity. All of the measurements were performed in a 400-μl fluorimetric quartz cuvette at 4.5 °C in a final sample volume of 300 μl of buffer containing 100 mm MOPS, pH 7.5, 180 mm NaCl, 3.5% glycerol, 0.1% 1,2-dioleoyl-sn-glycero-3-phosphocholine, 0.28% CHAPS, 0.4% bovine serum albumin (standard buffer), and 10 nm 5(6)-carboxyfluorescein-labeled proFIX. After measuring fluorescence anisotropy, we converted the values to the fraction of enzyme bound. The Kd values of proFIX for carboxylase were estimated as described previously (18Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). For titration of Na2S4O6-inactivated intact and trypsin-digested carboxylase, 5(6)-carboxyfluorescein-labeled proCon was used as ligand. Identification of AMS-labeled Cysteine Residue of Carboxylase—Carboxylase or Na2S4O6-inactivated carboxylase were incubated with 1 mm AMS on ice for 20 min. The reactions were stopped by DTT. AMS-labeled and nonlabeled carboxylase samples were subjected to SDS-PAGE. The protein bands were fixed by soaking the gel in 25% isopropanol with 10% acetic acid for 30 min, stained with 0.01% Coomassie R-250 in 10% acetic acid for 1 h, and destained by 10% acetic acid. The carboxylase bands were excised, and in-gel deglycosylation and trypsin digestion were performed as described previously (13Tie J.K. Mutucumarana V.P. Straight D.L. Carrick K.L. Pope R.M. Stafford D.W. J. Biol. Chem. 2003; 278: 45468-45475Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Kuster B. Mann M. Anal. Chem. 1999; 71: 1431-1440Crossref PubMed Scopus (140) Google Scholar). Trypsin digestion reaction supernatants were directly pooled for MALDI-TOF mass spectrometry (13Tie J.K. Mutucumarana V.P. Straight D.L. Carrick K.L. Pope R.M. Stafford D.W. J. Biol. Chem. 2003; 278: 45468-45475Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The digested sample (0.3 μl) and 0.3 μl of 10 mg/ml 1-cyano-4-hydroxysuccinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid (v/v) were deposited on the target plate and air-dried. MALDI mass spectra were recorded with an ABI 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA) MALDI-TOF/TOF mass spectrometer as described (13Tie J.K. Mutucumarana V.P. Straight D.L. Carrick K.L. Pope R.M. Stafford D.W. J. Biol. Chem. 2003; 278: 45468-45475Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). MALDI-TOF data from tryptic digests were calibrated with autoproteolytic peaks (internal standards), and mass errors were less than 20 ppm. TOF MS/MS scan functions were calibrated externally against the fragments of either angiotensin I or adrenocorticotropic hormone fragment 18–39 depending upon the precursor mass. The mass accuracy of collision-induced dissociation data was typically better than 50 ppm. Identification of the Last Transmembrane Domain of Carboxylase— Freshly prepared carboxylase-containing microsomes from High Five cells were digested by protease K as described previously (20Tie J. Wu S.M. Jin D. Nicchitta C.V. Stafford D.W. Blood. 2000; 96: 973-978Crossref PubMed Google Scholar). The reaction was stopped by the addition of phenylmethylsulfonyl fluoride (final concentration, 3 mm). The protease should degrade all of the peptide loops of carboxylase on the outside of the microsomal membrane, leaving the sequences inside the membranes intact. The microsomes were washed 3 times with the digestion buffer containing 3 mm phenylmethylsulfonyl fluoride. According to our previously published membrane topology (20Tie J. Wu S.M. Jin D. Nicchitta C.V. Stafford D.W. Blood. 2000; 96: 973-978Crossref PubMed Google Scholar), the C-terminal fragment with the last transmembrane domain of carboxylase should contain the HPC4 antibody recognition sequence, so we purified this fragment by affinity chromatography using the antibody column described before (20Tie J. Wu S.M. Jin D. Nicchitta C.V. Stafford D.W. Blood. 2000; 96: 973-978Crossref PubMed Google Scholar). The fraction bound to and eluted from the affinity chromatography column was concentrated by 10% trichloroacetic acid precipitation and fractionated by SDS-PAGE. The protein was transferred to a polyvinylidene fluoride membrane, and the C-terminal fragment was identified by Western blot using the HPC4 antibody. The N-terminal sequence of the peptide was determined at the Harvard Microchemistry Facility. Site-directed Mutagenesis of Carboxylase and Expression and Purification of Carboxylase in Insect Cells—Oligonucleotides and PCR primers used for mutagenesis were synthesized by Invitrogen. Site-directed mutagenesis of cysteines of human γ-glutamyl carboxylase was conducted by the "Megaprimer" method of PCR mutagenesis (21Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar, 22Datta A.K. Nucleic Acids Res. 1995; 23: 4530-4531Crossref PubMed Scopus (100) Google Scholar). Wild type carboxylase cDNA with a HPC4 tag at the C terminus (23Stearns 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) was used as template DNA for PCR. The mutations were screened by restriction digestion and verified by sequencing the entire cDNA. The wild type and cysteine mutants of carboxylase were expressed in High Five cells and purified as described previously (16Stanley 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). DTT was excluded from all of the purification steps, and the protease inhibition mixture was omitted from the sample destined for trypsin digestion. The active enzyme concentration was determined by fluorescence anisotropy titration of the enzyme against fluorescein-labeled consensus propeptide as described (18Presnell S.R. Tripathy A. Lentz B.R. Jin D.Y. Stafford D.W. Biochemistry. 2001; 40: 11723-11733Crossref PubMed Scopus (28) Google Scholar). Inactivation of Carboxylase by Thiol-reactive Reagents—Because vitamin K-dependent carboxylase is inactivated by a variety of thiol-reactive reagents, free cysteines have been implicated as comprising part of the carboxylase active site (3Morris D.P. Soute B.A. Vermeer C. Stafford D.W. J. Biol. Chem. 1993; 268: 8735-8742Abstract Full Text PDF PubMed Google Scholar, 8Canfield L.M. Sinsky T.A. Suttie J.W. Arch. Biochem. Biophys. 1980; 202: 515-524Crossref PubMed Scopus (25) Google Scholar, 9Canfield L.M. Biochem. Biophys. Res. Commun. 1987; 148: 184-191Crossref PubMed Scopus (19) Google Scholar, 10Bouchard B.A. Furie B. Furie B.C. Biochemistry. 1999; 38: 9517-9523Crossref PubMed Scopus (19) Google Scholar). Fig. 1 shows the inactivation curve of carboxylase by increasing concentrations of different thiol-reactive reagents at a fixed reaction time of 20 min at 0 °C. Na2S4O6 and AMS (a hydrophilic maleimide derivative) inactivate carboxylase. In contrast, the hydrophobic maleimide derivative DABMI, which has a structure similar to AMS, does not inactivate carboxylase at concentrations up to 5 mm. These results suggest that cysteine residues that affect the enzyme activity by chemical modification may be located in the hydrophilic but not the hydrophobic regions of the carboxylase. Hydrophilic Maleimide and Na2S4O6 Modify the Same Cysteine Residues of Carboxylase—We next examined whether Na2S4O6 and AMS inhibit carboxylase activity by reacting with the same cysteine residue(s). To accomplish this, carboxylase was first treated with Na2S4O6, whose modification can be reversed by DTT. This tetrathionated enzyme was then treated with AMS. More than 90% of the carboxylase activity of Na2S4O6-inactivated carboxylase was recovered after DTT treatment (Fig. 2). AMS-modified carboxylase is irreversibly inactivated. However, carboxylase first modified by Na2S4O6 and then treated with AMS regains about 80% of its activity following DTT treatment. These results suggest that AMS and Na2S4O6 modify the same cysteine residue(s) of the carboxylase. Protection of Na2S4O6 Inactivation of Carboxylase—If cysteine residues are part of the active site, then one might expect substrates to protect the enzyme from inactivation by thiol-reactive reagents. In addition to the glutamate substrate, γ-carboxylation requires VKH2, carbon dioxide, and oxygen as co-substrates. Moreover, the propeptide of the Glu-containing substrate stimulates carboxylation (24Knobloch J.E. Suttie J.W. J. Biol. Chem. 1987; 262: 15334-15337Abstract Full Text PDF PubMed Google Scholar). Our results (Table I) show that proFIX, proFIXgla, VKH2, pentapeptide substrate (FLEEL), and different combinations of these substrates protect the carboxylase from inactivation by Na2S4O6 to various extents.Table IProtection of carboxylase from inactivation by Na2S4O6SubstrateActivity (means ± S.D., n = 3)%Blank43 ± 1proFIX80 ± 1proFIX/FLEEL81 ± 2proFIX/VKH289 ± 1proFIX/VKH2/FLEEL86 ± 1proFIXgla91 ± 1proFIXgla / VKH293 ± 2FLEEL42 ± 1VKH266 ± 1VKH2/FLEEL72 ± 1 Open table in a new tab It is noteworthy that proFIX and proFIXgla both significantly protect carboxylase from inactivation by Na2S4O6, but carboxylase that has been nicked at residues 349 and 351 by limited trypsin digestion is less well protected, although still active. After 1 h of incubation with Na2S4O6, intact carboxylase retains about 30% activity in the absence of proFIX, whereas in the presence of proFIX about 80% activity remains (Fig. 3A). In contrast, the "nicked" carboxylase is not protected by the propeptide (Fig. 3B). Propeptide Binding Capacity of Intact and Nicked Carboxylase—One possible reason the nicked enzyme was not protected from Na2S4O6 inactivation may be because it does not bind propeptide. To test this we compared the propeptide binding of the nicked enzyme to that of intact carboxylase using fluoroscein-labeled proFIX. Our results show (Fig. 4A) that the nicked carboxylase has binding characteristics similar to those of the intact carboxylase. The Kd values for proFIX/intact carboxylase and proFIX/nicked carboxylase are 16.3 ± 0.4 and 37.3 ± 1nm, respectively. Because we used a high concentration of propeptide in the protection experiments (8 μm), the enzyme should be saturated with substrate, and the loss of protection of propeptide against Na2S4O6 inactivation of nicked carboxylase is not due to a lack of propeptide binding to the nicked enzyme. These results suggest that Na2S4O6-modified cysteine residues are not located in the propeptide-binding site. We further examined the propeptide binding capacities of Na2S4O6-inactivated carboxylase using the fluoroscein-labeled proCon. As shown in Fig. 4B, modification by Na2S4O6 significantly decreases the affinity of carboxylase for the consensus propeptide (Kd = 35 versus 0.3 nm for unmodified). We observed a similar, although less dramatic, effect on the trypsin-nicked modified enzyme (Fig. 4B). Identification of AMS-modified Cysteine Residues of Carboxylase by MALDI-TOF MS—We employed MALDI-TOF MS to identify cysteine residues modified by the hydrophilic thiol-reactive reagent, AMS. The spectra for trypsin-digested modified and nonmodified samples are shown in Figs. 5 and 6. Compared with the nonmodified sample (Fig. 5, A, C, and E, and Fig. 6, A and C), we identified five major new peptide peaks (m/z at 1603.63, 2409.13, 2537.22, 1915.77, and 2849.37) in the spectra of the AMS-modified sample (Fig. 5, B, D, and F, and Fig. 6, B and D). The new peptide peaks at m/z 1603.63, 2409.13, and 2537.22 (Fig. 5, B, D, and F) represent AMS modification of Cys323 containing tryptic peptides, KLVSYCPQR (m/z at 1093.58), LVSYCPQRLQQLLPLK (m/z at 1899.09), and KLVSYCPQRLQQLLPLK (m/z at 2027.18), respectively. The peptide peaks at m/z 1915.77 and 2849.37 (Fig. 6, B and D) derive from AMS modification of Cys343-containing tryptic peptides AAPQPSVSCVYKR (m/z at 1405.73) and LQQLLPLKAAPQPSVSCVYKR(m/z at 2339.33), respectively. The skipped trypsin cleavage sites are identified in bold type. All 10 cysteines of the carboxylase were identified, and Cys323 and Cys343 are the only two cysteines modified by AMS under our reaction conditions. The mass increase from AMS modifica
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