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• W2079475368 abstract "Two succinyl-CoA synthetases, one highly specific for GTP/GDP and the other for ATP/ADP, have been purified to homogeneity from pigeon liver and breast muscle. The two enzymes are differentially distributed in pigeon, with only the GTP-specific enzyme detected in liver and the ATP-specific enzyme in breast muscle. Based on assays in the direction of CoA formation, the ratios of GTP-specific to ATP-specific activities in kidney, brain, and heart are ∼7, 1, and 0.1, respectively. Both enzymes have the characteristic α- and β-subunits found in other succinyl-CoA synthetases. Studies of the α-subunit by electrophoresis, mass spectrometry, reversed-phase high performance liquid chromatography, and peptide mapping showed that it was the same in the two enzymes. Characterization of the β-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the β-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same α-subunit, the determinants of nucleotide specificity must reside within the β-subunit. Determination of the apparent Michaelis constants showed that the affinity of the GTP-specific enzyme for GDP is greater than that of the ATP-specific enzyme for ADP (7 versus 250 μm). Rather large differences in apparentK m values were also observed for succinate and phosphate. Two succinyl-CoA synthetases, one highly specific for GTP/GDP and the other for ATP/ADP, have been purified to homogeneity from pigeon liver and breast muscle. The two enzymes are differentially distributed in pigeon, with only the GTP-specific enzyme detected in liver and the ATP-specific enzyme in breast muscle. Based on assays in the direction of CoA formation, the ratios of GTP-specific to ATP-specific activities in kidney, brain, and heart are ∼7, 1, and 0.1, respectively. Both enzymes have the characteristic α- and β-subunits found in other succinyl-CoA synthetases. Studies of the α-subunit by electrophoresis, mass spectrometry, reversed-phase high performance liquid chromatography, and peptide mapping showed that it was the same in the two enzymes. Characterization of the β-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the β-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same α-subunit, the determinants of nucleotide specificity must reside within the β-subunit. Determination of the apparent Michaelis constants showed that the affinity of the GTP-specific enzyme for GDP is greater than that of the ATP-specific enzyme for ADP (7 versus 250 μm). Rather large differences in apparentK m values were also observed for succinate and phosphate. succinyl-CoA synthetase GTP-specific SCS ATP-specific SCS high performance liquid chromatography 4-morpholinepropanesulfonic acid nucleoside diphosphate nucleoside triphosphate polyacrylamide gel electrophoresis. Succinyl-CoA synthetase (SCS1; also known as succinate thiokinase) participates in the Krebs cycle, where it catalyzes a substrate-level phosphorylation of GDP or ADP. Succinyl-CoA synthetase may also catalyze the reverse reaction to supply succinyl-CoA for heme synthesis and ketone body activation (1Labbe R.F. Kurumada T. Onisawa J. Biochim. Biophys. Acta. 1965; 111: 403-415Crossref PubMed Scopus (36) Google Scholar, 2Ottaway J.H. McClellan J.A. Saunderson C.L. Int. J. Biochem. 1981; 13: 401-410Crossref PubMed Scopus (44) Google Scholar). Both GTP-specific (EC 6.2.1.4) and ATP-specific (EC 6.2.1.5) forms of SCS are known. For ∼20 years following the discovery of SCS in the early 1950s, there was evidence for the occurrence of only G-SCS in animals. Only A-SCS has been found in plants, whereas bacterial enzymes tend to be nonspecific, although ones strongly preferring ATP or GTP are known (3Nishimura J.S. Adv. Enzymol. Relat. Areas Mol. Biol. 1986; 58: 141-172PubMed Google Scholar,4Bridger W.A. Boyer P.D. The Enzymes. X. Academic Press, New York1974: 581-606Google Scholar). Beginning in the early 1970s, a variety of evidence has accumulated indicating that A-SCS is also found in animals. This evidence includes partial purification of A-SCS from blowfly (5Hansford R.G. FEBS Lett. 1973; 31: 317-320Crossref PubMed Scopus (16) Google Scholar) and pigeon breast muscle (6Allen D.A. Ottaway J.H. FEBS Lett. 1986; 194: 171-175Crossref PubMed Scopus (10) Google Scholar). Assays of extracts showed that adenine and guanine nucleotides support SCS activity to varying degrees in tissue preparations from pigeon and chicken (7Hamilton M.L. Ottaway J.H. FEBS Lett. 1981; 123: 252-254Crossref PubMed Scopus (14) Google Scholar), several mammalian species (8Weitzman P.D.J. Jenkins T. Else A.J. Holt R.A. FEBS Lett. 1986; 199: 57-60Crossref PubMed Scopus (18) Google Scholar), and a wide variety of animals including insects and crustaceans (9McClellan J.A. Ottaway J.H. Comp. Biochem. Physiol. B Comp. Biochem. 1980; 67: 679-684Crossref Scopus (13) Google Scholar). Thus, there is evidence that both isoforms are present in some animals/tissues, whereas only A-SCS or G-SCS may be found in others. A study of the inhibition of SCS activity by vanadate indicated two forms of the enzyme (10Krivanek J. Novakova L. Physiol. Res. 1992; 41: 345-350PubMed Google Scholar). Steiner and Smith (11Steiner A.W. Smith R.A. J. Neurochem. 1981; 37: 582-593Crossref PubMed Scopus (19) Google Scholar) observed that the α-subunit of SCS in rat brain is more readily labeled by ATP than by GTP, whereas that in rat liver mitoplasts is labeled only by GTP. The possibility that A-SCS and G-SCS serve different roles has received attention. McClellan and Ottaway (9McClellan J.A. Ottaway J.H. Comp. Biochem. Physiol. B Comp. Biochem. 1980; 67: 679-684Crossref Scopus (13) Google Scholar) reported a correlation of the presence of G-SCS in muscle with the utilization of ketone bodies. Based on changes in the ratio of G-SCS to A-SCS during experimentally induced diabetes and porphyria, Jenkins and Weitzman (12Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1986; 205: 215-218Crossref PubMed Scopus (22) Google Scholar, 13Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1988; 230: 6-8Crossref PubMed Scopus (12) Google Scholar) proposed that A-SCS participates in the Krebs cycle, whereas G-SCS catalyzes the reverse reaction in support of heme synthesis and ketone body activation. All SCSs characterized to date have α- and β-types of subunits. In Gram-negative bacteria, the enzyme has an α2β2-tetrameric structure, whereas in other bacteria (14Weitzman P.D.J. Kinghorn H.A. FEBS Lett. 1978; 88: 255-258Crossref PubMed Scopus (26) Google Scholar) and higher organisms, only the αβ-heterodimer has been found. The α-subunit possesses the histidine residue that is phosphorylated as a part of the catalytic cycle (15Bridger W.A. Biochem. Biophys. Res. Commun. 1971; 42: 948-954Crossref PubMed Scopus (36) Google Scholar). Two forms of G-SCS generated by alternative splicing of the α-subunit have been found in pig heart (16Ryan D.G. Lin T. Brownie E. Bridger W.A. Wolodko W.T. J. Biol. Chem. 1997; 272: 21151-21159Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Although evidence varies as to the roles of the α- and β-subunits in binding substrates, it seems clear that the catalytic site is shared between the subunits (3Nishimura J.S. Adv. Enzymol. Relat. Areas Mol. Biol. 1986; 58: 141-172PubMed Google Scholar). An x-ray crystallographic study of the Escherichia coli enzyme provides a framework for further structure-function studies (17Wolodko W.T. Fraser M.E. James M.N.G. Bridger W.A. J. Biol. Chem. 1994; 269: 10883-10890Abstract Full Text PDF PubMed Google Scholar). In the work presented here, we demonstrate by enzyme purification and characterization that highly ATP- and GTP-specific forms of SCS occur in pigeon. We further show that these two forms incorporate the same α-subunit. The following paper (18Johnson J.D. Mehus J.G. Tews K. Milavetz B.I. Lambeth D.O. J. Biol. Chem. 1998; 273: 27580-27586Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) presents gene sequences for both forms and provides unequivocal evidence that A-SCS, as well as G-SCS, is expressed in mammalian species. Chromatographic materials were obtained as follows: QAE-Sepharose, DEAE-Sephacel, and AMP-Sepharose 4B (ligand attached through the N 6-amino group) from Sigma; Bio-Gel HT and Affi-Gel blue from Bio-Rad; P-11 cellulose phosphate from Whatman; a 4.6 × 250-mm Microsorb-MV C-18 HPLC column from Rainin Instrument Co. Inc.; and a 4.6 × 250-mm Ultrasphere IP 5μ C-18 HPLC column from Beckman Instruments. Reagents used were the highest grade available from Sigma or Fisher, unless noted otherwise. Succinyl-CoA was synthesized as described by Simon and Shemin (19Simon E.J. Shemin D. J. Am. Chem. Soc. 1953; 75: 2520Crossref Scopus (538) Google Scholar) and was stored at −20 °C at pH <2. All solutions were prepared in water purified by the Milli-Q water purification system from Millipore Corp. Pigeons used for enzyme purification and assays were free-ranging birds of both sexes and were obtained from a local breeder. All purification procedures for either enzyme were carried out at 0–4 °C. Livers from 12 pigeons were finely minced with scissors and homogenized with a Potter-Elvehjem homogenizer using 600 ml of homogenization medium (0.22 mmannitol, 0.070 m sucrose, and 0.020 m MOPS). The particulate fraction obtained by centrifugation for 10 min at 40,000 × g was washed repeatedly by resuspension and centrifugation until the supernatant was clear. The pellet from the final isotonic wash was suspended in 20 mm phosphate, pH 7.6, containing 1 mm EDTA and 1 mmdithiothreitol and centrifuged. The resulting pellet was resuspended in 250 ml of the phosphate buffer and stored at −70 °C. The GTP-specific enzyme was purified to homogeneity by a series of chromatography steps. Unless stated otherwise, all buffers were adjusted to pH 7.8 and contained 1 mm dithiothreitol and 1 mm EDTA. Phosphate buffers were prepared from their monopotassium salt and adjusted to the desired pH with KOH. The frozen particulate fraction derived from 12 livers was thawed and then sonicated in 80-ml batches using a Branson sonifier 250. Sonication was carried out for two periods of 30 s each, with a 45-s pause between. The sonicated suspension was centrifuged for 60 min at 40,000 × g. The supernatant was filtered through cheesecloth and then diluted 2-fold with 5 mm phosphate. The filtrate was chromatographed on a 5 × 11-cm QAE-Sepharose column equilibrated with 13 mm phosphate buffer. Prolonged washing of the column with the equilibration buffer resulted in G-SCS activity eluting considerably after the breakthrough peak. The pool of SCS activity from QAE-Sepharose was added to a 2.5 × 14-cm Affi-Gel blue column and washed with 5 bed volumes of 13 mm phosphate buffer, 5 volumes of 850 mmphosphate buffer, and 5 volumes of 20 mm phosphate. The enzyme was eluted with 1 m KCl and 20 mmphosphate. The pool from the Affi-Gel blue column was added to a 1.5 × 9.5-cm hydroxylapatite (Bio-Gel HT) column and washed with 1 m KCl and 20 mm phosphate until the enzyme eluted, which came after a major contaminating peak. The eluted enzyme was dialyzed for 8 h against 4 liters of 100 mm phosphate. Ammonium sulfate was added (0.53 g/ml), and the resulting suspension was stored at 4 °C. Enzyme purification was continued after pelleting the ammonium sulfate suspension by centrifugation for 40 min at 40,000 × g. The pellet was dissolved in 1.5 ml of 25 mm phosphate buffer, pH 6.7, and equilibrated with this buffer by chromatography on a 1.5 × 25-cm column of Sephadex G-25. The active fractions were combined and added to a 0.7 × 8-cm cellulose phosphate column equilibrated with the above buffer. The column was washed with 5 volumes of 25 mm phosphate buffer, pH 6.7. The enzyme was eluted using 50 mm phosphate, pH 6.7, and then concentrated with a Centricon-30 (Amicon, Inc.). The purified enzyme was stable when stored at −70 °C. The buffers used for purification of A-SCS differed from those used for G-SCS in that they were adjusted, unless otherwise noted, to pH 7.6, and dithiothreitol and EDTA were omitted. Breast muscle from three adult pigeons was homogenized in 1.2 liters of homogenization buffer using a Waring blender operated at maximum power for 1 min. The pellet obtained after centrifugation for 10 min at 15,000 × g was washed by repeated suspension in homogenization buffer and centrifugation until the supernatant was clear. The washed pellet was resuspended in 20 mm potassium phosphate buffer, pH 7.6, and centrifuged. The resulting pellet was resuspended in 600 ml of the same phosphate buffer and stored at −70 °C. An extract was prepared from the thawed particulate fraction in the same manner as described above for G-SCS, except the buffer concentration was maintained at 20 mm phosphate. The extract was added to a 5 × 11.5-cm DEAE-Sephacel column with the flow-through fraction directed to a 5 × 10.5-cm hydroxylapatite (Bio-Gel HT) column. The DEAE column served to filter out interfering lipids. The columns were washed with 5 volumes of 20 mmphosphate before disconnecting the DEAE column. The hydroxylapatite column was then washed with 5 volumes of 1 m KCl and 20 mm phosphate, followed by 5 volumes of 20 mmphosphate. The enzyme was eluted with 400 mm phosphate, pH 7.6. The enzyme pool from chromatography on hydroxylapatite was added to a 2.5 × 12.5-cm column of Affi-Gel blue that had been equilibrated with 400 mm phosphate. This column was then washed with 3 volumes of 400 mm phosphate buffer, 5 volumes of 850 mm phosphate buffer, and 5 volumes of 20 mmphosphate buffer, all at pH 7.6. The enzyme was eluted with 1m KCl and 20 mm phosphate. The enzyme was concentrated to <30 ml in an Amicon concentrator equipped with a YM-30 membrane and rechromatographed on a 1.5 × 12-cm column of hydroxylapatite. This column was washed with 5 volumes of 1 m KCl and 20 mm phosphate and 5 volumes of 20 mm phosphate, pH 7.6. The enzyme was then eluted with 400 mm phosphate, pH 7.6. Rechromatography on hydroxylapatite further purified and concentrated the enzyme and also separated it from KCl, which forms a precipitate in the ammonium sulfate fractionation step. The enzyme was precipitated with ammonium sulfate and divided into three aliquots for storage. To continue the purification, an aliquot was desalted on Sephadex G-25 equilibrated with 10 mmphosphate buffer, pH 6.7. The active fractions were combined and added to a 0.7 × 8-cm adenosine 5′-monophosphate-Sepharose 4B column equilibrated with 10 mm phosphate, pH 6.7. The A-SCS activity appeared in the flow-through fractions, which were collected and added to a 1 × 3.2-cm cellulose phosphate column equilibrated with 10 mm phosphate, pH 6.7. This column was washed with 5 volumes of 10 mm phosphate buffer, pH 6.7, followed by 5 volumes of 80 mm phosphate, pH 6.7. The enzyme was eluted with 125 mm phosphate, pH 6.7. Active fractions were concentrated using a Centricon-30. The sample was stable when stored frozen at −70 °C in phosphate buffer. HPLC-based assays were conducted as described previously (20Lambeth D.O. Muhonen W.W. Anal. Biochem. 1993; 209: 192-198Crossref PubMed Scopus (20) Google Scholar) and were used during purification procedures and to determine nucleotide specificity. Briefly, assays proceeded for 5 min at 30 °C and pH 7.6 in a final volume of 0.3 ml containing 50 mm HEPES, 50 mm phosphate, 10 mmMgCl2, 1 mm ADP (or GDP), and 1 mmsuccinyl-CoA. Succinyl-CoA was added immediately preceding a 5-min preincubation, and assays were initiated by adding the enzyme. Sufficient KOH was included in each assay to neutralize the HCl in the succinyl-CoA preparation. Reactions were stopped by adding 0.35 ml of 1m formic acid. The percentage conversion of succinyl-CoA to CoA was determined by HPLC using a Beckman C-18 column (20Lambeth D.O. Muhonen W.W. Anal. Biochem. 1993; 209: 192-198Crossref PubMed Scopus (20) Google Scholar). Two controls, one lacking enzyme and the other ADP (or GDP), were run in parallel to correct for spontaneous hydrolysis of succinyl-CoA and succinyl-CoA hydrolase activity. The latter activity was always <10% of SCS activity and was eliminated in the initial chromatography steps. Continuous spectrophotometric assays were used in kinetic studies. In the direction of ADP (GDP) formation, the standard assay contained 100 mm Tris acetate, pH 8.0, 0.1 mm CoA, 1.0 mm ATP (or GTP), 10 mm succinate, 7.1 mm magnesium acetate, 10 mm KCl, 1.55 mm phosphoenolpyruvate, 0.1 mm NADH, 1.5 units of pyruvate kinase, and 1.5 units of lactate dehydrogenase. Phosphate was removed from stocks of A-SCS or G-SCS by chromatography on Sephadex G-25 equilibrated with 25 mm Tris succinate, pH 7.6. Reactions were initiated by adding succinyl-CoA synthetase and were carried out at 30 °C in 200-μl microcuvettes in a Beckman Model 640 spectrophotometer. When assaying A-SCS in the direction of ATP synthesis, the standard assay contained 0.1 m Tris acetate, pH 8.0, 0.1 mm succinyl-CoA, 1.0 mm ADP, 10 mmphosphate. 7.1 mm magnesium acetate, 10 mm KCl, 1 mm glucose, 0.1 mm NADP, 1 unit of hexokinase, and 1 unit of glucose-6-phosphate dehydrogenase. Reactions were initiated by adding SCS. To assay G-SCS, the standard assay substituted 1.0 mm GDP for 1.0 mm ADP, but also contained 2 units of NDP kinase and 0.05 mm ADP in order to obtain coupling to the hexokinase/glucose-6-phosphate dehydrogenase system. Prior experiments had shown that ADP does not function as a substrate for G-SCS. The apparent K m for each substrate was determined by obtaining at least two sets of velocity versus concentration data. A minimum of six concentrations were used in each set. Each substrate was varied at fixed (i.e. “standard”) concentrations of the other two substrates. The velocities obtained were numerically fitted by reiteration to the Michaelis-Menten equation (v =V maxS/(K m + S)). When substrate inhibition was observed (GDP, GTP, and ATP as variable substrate), the data were fitted to a modified form of the Michaelis-Menten equation (v =V maxS/(K m(1 + I/K iS) + S)), whereK iS is the inhibition constant for the substrate. Data points were not weighted. The protein concentration at various stages of purification of each SCS enzyme was estimated using the value of A 2801 mg/ml = 0.350, as reported for pig heart SCS (21Murakami Y. Nishimura J.S. Biochim. Biophys. Acta. 1974; 336: 252-263Crossref Scopus (14) Google Scholar). Purity and molecular masses of the subunits of each enzyme were determined on 18% gels by SDS-PAGE. The isoelectric points of the holoenzymes were estimated on a pH 3–10 gel from Novex (San Diego, CA) using the isoelectric focusing standards kit from Bio-Rad. The positions of the focused proteins were determined by staining with Coomassie Blue. The subunits of each SCS enzyme were separated and purified by reversed-phase chromatography on a Rainin Microsorb-MC C-18 column. Approximately 25 μg of enzyme was injected onto a 4 × 250-mm column. A linear gradient over a period of 22 min was run from 0.1% trifluoroacetic acid in water to a mixture containing 98% acetonitrile, 1.9% water, and 0.1% trifluoroacetic acid. All solvent components were HPLC-grade. The eluted polypeptides were detected by their absorbance at 220 nm. This technique was used to purify sufficient amounts of each subunit for tryptic peptide mapping and isoelectric focusing. Fractions containing peptide were evaporated to dryness and redissolved in the desired buffer. Tryptic peptide maps were prepared by the Harvard Microchemistry Facility. Subunits purified by HPLC were concentrated and then further purified by SDS-PAGE. The bands were subjected to in-gel tryptic digestion as described by Hellman et al. (22Hellman U. Wernstedt C. Gonez J. Heldin C.-H. Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (686) Google Scholar), but without the addition of 0.02% Tween. The resulting peptide mixture was separated by microbore high performance liquid chromatography using a Zorbax C-18 1.0-mm × 150-mm reversed-phase column and a Hewlett-Packard 1090 HPLC/1040 diode array detector. Peptides were detected by differential UV absorbance at 205, 277, and 292 nm. The masses of the α-subunits of purified A-SCS and G-SCS were determined by electrospray mass spectrometry after separation of the subunits by HPLC. The results for purification of A-SCS from breast muscle and G-SCS from liver are summarized in Tables I and II, and the procedures are described under “Experimental Procedures.” Both enzymes were purified from particulate fractions containing mitochondria that had been subjected to hypotonic shock, freeze-thawing, and sonication. The purification schemes include chromatographies on hydroxylapatite and Affi-Gel blue, for which binding and elution of enzyme are not a straightforward function of ionic strength. Final purification of both enzymes was achieved by chromatography on cellulose phosphate. This column was particularly effective in removing a major contaminant that co-migrated with the β-subunit of A-SCS during SDS-PAGE.Table IPurification of GTP-specific SCS from pigeon liverStepVolumeEnzyme unitsProteinSpecific activityRecoveryPurificationmlμmol/minmgunits/mg%-foldExtract21515116,7000.0091001QAE-Sepharose44513151870.025873.2Affi-Gel blue6162.81100.5741.763.3Hydroxylapatite6036.359.50.6124.167.8Ammonium sulfate fractionation12.510.42.54.166.91462.2Phosphocellulose135.9.2722.13.922456The enzyme was purified from an extract of the particulate fraction obtained from the livers of 12 adult pigeons. Details of the methods are presented under “Experimental Procedures.” Open table in a new tab Table IIPurification of ATP-specific SCS from pigeon breast muscleStepVolumeEnzyme unitsProteinSpecific activityRecoveryPurificationmlμmol/minmgunits/mg%-foldExtract42044035,0400.01261001DEAE-Sephacel/Hydroxylapatite15830823500.1317010.4Affi-Gel blue1401682160.7783861.7Hydroxylapatite171421480.9593276.1Ammonium sulfate fractionation2-aThe last three rows were extrapolated from the data obtained by taking one-third quantities of the batch from the hydroxylapatite step through the purification procedure.33.6101.71430.7123.156.4AMP-Sepharose 4B8770.244.41.5916.0126Phosphocellulose4851.67.836.6111.8525The enzyme was purified from an extract of the particulate fraction obtained from the breast muscle of three adult pigeons. Details of the methods are presented under “Experimental Procedures.”2-a The last three rows were extrapolated from the data obtained by taking one-third quantities of the batch from the hydroxylapatite step through the purification procedure. Open table in a new tab The enzyme was purified from an extract of the particulate fraction obtained from the livers of 12 adult pigeons. Details of the methods are presented under “Experimental Procedures.” The enzyme was purified from an extract of the particulate fraction obtained from the breast muscle of three adult pigeons. Details of the methods are presented under “Experimental Procedures.” Analysis by SDS-PAGE showed that highly purified A-SCS and G-SCS contained two types of subunits, α and β (Fig. 1), which is characteristic of all other SCSs studied to date. The α-subunit of each enzyme migrated with the same mobility on SDS-PAGE, whereas the β-subunit of A-SCS migrated slightly faster than that of G-SCS. The β-subunits separated when a mixture of the two purified enzymes was electrophoresed (data not shown). The apparent molecular masses were estimated to be ∼35,000 Da for the α-subunit and 42,500 and 44,500 Da for the β-chains of A-SCS and G-SCS, respectively. Purified preparations of native A-SCS and G-SCS showed apparent pI values of ∼7.9 and 7.3, respectively, when isoelectrically focused (data not shown). The results of focusing each subunit purified by HPLC indicated that the pI of the α-subunits of A-SCS and G-SCS was the same, ∼9. Both β-subunits focused in the range of 5–6, with the apparent pI of the β-subunit of A-SCS being slightly higher than that of the β-subunit of G-SCS. Purified preparations of both enzymes were chromatographed on a Rainin reversed-phase column as described under “Experimental Procedures.” As shown in Fig. 2, the α-subunits of the two enzymes migrated with the same mobility, and each α-subunit eluted before its respective β-subunit. The identity of each peak was confirmed by analysis of the eluted polypeptide by SDS-PAGE. When a mixture of the two enzymes was chromatographed, the α-subunits gave a single symmetrical peak, whereas the β-subunits were well resolved (data not shown). The subunits of each enzyme were separated and purified by HPLC before digestion with trypsin. The tryptic maps of the α-subunits, shown in Fig. 3(A and C), are virtually identical, further indicating that the same α-subunit is incorporated into A-SCS and G-SCS. In contrast, the tryptic peptide maps of the β-subunits of A-SCS and G-SCS (Fig. 3, B and D) are very different. This is consistent with the peptides being encoded by different genes rather than being generated by alternative splicing. The masses of the α-subunits of purified A-SCS and G-SCS were determined by mass spectrometry to be 32,170 ± 22 and 32,198 ± 29 Da, respectively. These values are consistent with the same α-subunit being incorporated into A-SCS and G-SCS. The specificity of each purified enzyme for its nucleotide was determined using HPLC-based assays that measured the NDP-supported conversion of succinyl-CoA to CoA (20Lambeth D.O. Muhonen W.W. Anal. Biochem. 1993; 209: 192-198Crossref PubMed Scopus (20) Google Scholar). The use of GDP by the enzyme purified from breast muscle (A-SCS) was negligible even when its concentration was increased to 5 mm (data not shown). Similarly, the liver enzyme (G-SCS) showed only a small apparent reaction with ADP (<1% of the rate with GDP). The apparent kinetic constants of the two enzymes were determined by continuous enzyme-coupled assays. Both directions of catalysis were studied as described under “Experimental Procedures.” The concentration of each substrate was varied around its apparent K m, whereas the concentrations of the other two substrates were fixed at their standard values (Tables III and IV). The apparent K mvalues were determined by numerically fitting initial velocities as a function of substrate concentration to the Michaelis-Menten equation. The apparent K m values obtained are shown in Tables III and IV. Except for the nucleoside di- and triphosphates, which showed substrate inhibition, the observed velocities conformed closely to the Michaelis-Menten equation.Table IIIApparent Michaelis constants in the direction of succinyl-CoA synthesisEnzymeNTPVaried substrateConcentrations of substratesApparentK mNTPSuccinateCoAmmmmA-SCSATPATP0.005–2.36100.10.055 ± 0.005Succinate11.0–100.15.1 ± 1.7CoA1100.005–0.0920.032 ± 0.007G-SCSGTPGTP0.004–1.94100.10.036 ± 0.008Succinate10.25–100.10.49 ± 0.07CoA1100.006–0.1140.036 ± 0.005 Open table in a new tab Table IVApparent Michaelis constants in the direction of succinate synthesisEnzymeNDPVaried substrateConcentrations of substratesApparentK mNDPSuccinyl-CoAPhosphatemmmmA-SCSADPADP0.004–1.580.1100.25 ± 0.02Succinyl-CoA10.004–0.086100.041 ± 0.008Phosphate10.10.25–100.72 ± 0.14G-SCSGDPGDP0.0014–1.390.1100.007 ± 0.0007Succinyl-CoA10.004–0.086100.086 ± 0.012Phosphate10.10.25–102.26 ± 0.39 Open table in a new tab When adenine or guanine nucleotide was the variable substrate, the initial velocities were fitted to a modified form of the Michaelis-Menten equation that allowed for substrate inhibition (see “Experimental Procedures”). Fig. 4shows plots of the data for ADP and GDP. As can be seen, GDP exhibited both a very low K m (∼7 μm) and very strong substrate inhibition. The two forms of SCS show very differentK m values for two substrates other than NDP. TheK m of A-SCS for succinate is ∼10-fold higher than that of G-SCS. In contrast, G-SCS shows a 3-fold higherK m for phosphate. In HPLC-based assays of unfractionated homogenates, A-SCS was not detected in liver tissue, nor G-SCS in breast muscle. The assays were conducted in the direction of CoA and NTP formation. Both activities were present in kidney, brain, and heart, with the ratio of activities being in substantial agreement with the data of Hamilton and Ottaway (7Hamilton M.L. Ottaway J.H. FEBS Lett. 1981; 123: 252-254Crossref PubMed Scopus (14) Google Scholar). Thus, as shown in Table V, most of the SCS activity in kidney is due to G-SCS, whereas A-SCS predominates in heart. Approximately equal amounts of the two activities are found in brain.Table VRatio of G-SCS to A-SCS in various pigeon tissuesTissueG-SCS/A-SCSMeasured5-aHPLC-based assays were used to quantify GDP- and ADP-supported, enzyme-catalyzed conversion of succinyl-CoA to succinate.Published5-bData were from Hamilton and Ottaway (7). These values were based on a spectrophotometric assay in the direction of succinyl-CoA formation.Heart0.110.21Brain0.900.45Kidney7.24.85-a HPLC-based assays were used to quantify GDP- and ADP-supported, enzyme-catalyzed conversion of succinyl-CoA to succinate.5-b Data were from Hamilton and Ottaway (7Hamilton M.L. Ottaway J.H. FEBS Lett. 1981; 123: 252-254Crossref PubMed Scopus (14) Google Scholar). These values were based on a spectrophotometric assay in the direction of succinyl-CoA formation. Open table in a new tab The data presented here confirm that highly ATP- and GTP-specific forms of SCS are present in pigeon and that the two forms are present in widely different ratios in the tissues assayed (7Hamilton M.L. Ottaway J.H. FEBS Lett. 1981; 123: 252-254Crossref PubMed Scopus (14) Google Scholar). Both enzymes contain α- and β-types of subunits, with several lines of experimental evidence indicating that the α-subunits are identical. This finding means that the determinants of nucleotide specificity must lie within the β-subunit. The peptide maps of the β-subunits of the two enzymes show extensive differences, which suggests that they are the products of different genes. The sequences of both forms of the β-subunit in pigeon and three mammalian species are reported in the accompanying paper (18Johnson J.D. Mehus J.G. Tews K. Milavetz B.I. Lambeth D.O. J. Biol. Chem. 1998; 273: 27580-27586Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Prior evidence regarding the location of the NDP/NTP-binding site has led to conflicting conclusions. The site has most frequently been suggested to lie within the α-subunit (3Nishimura J.S. Adv. Enzymol. Relat. Areas Mol. Biol. 1986; 58: 141-172PubMed Google Scholar, 23Majumdar R. Biochim. Biophys. Acta. 1991; 1076: 86-90Crossref PubMed Scopus (14) Google Scholar, 24Birney M.A. Klein C. Arch. Biochem. Biophys. 1995; 319: 93-101Crossref PubMed Scopus (13) Google Scholar) based on the observation (3Nishimura J.S. Adv. Enzymol. Relat. Areas Mol. Biol. 1986; 58: 141-172PubMed Google Scholar) that the isolated α-subunit retains the capacity to autophosphorylate in a reaction that is stimulated by the addition of the β-subunit (25Pearson P.H. Bridger W.A. J. Biol. Chem. 1975; 250: 8524-8529Abstract Full Text PDF PubMed Google Scholar). X-ray diffraction revealed three potential nucleotide-binding sites: two in the α-subunit and one in the β-subunit (17Wolodko W.T. Fraser M.E. James M.N.G. Bridger W.A. J. Biol. Chem. 1994; 269: 10883-10890Abstract Full Text PDF PubMed Google Scholar). The presence of CoA in the N-terminal domain of the α-subunit eliminated that site as the one binding ADP/ATP; prior evidence had indicated that the CoA-binding site was in the β-subunit (23Majumdar R. Biochim. Biophys. Acta. 1991; 1076: 86-90Crossref PubMed Scopus (14) Google Scholar, 26Collier G.E. Nishimura J.S. J. Biol. Chem. 1978; 253: 4938-4943Abstract Full Text PDF PubMed Google Scholar, 27Buck D. Guest J.R. Biochem. J. 1989; 260: 737-747Crossref PubMed Scopus (32) Google Scholar). Data indicating that the NDP/NTP-binding site lies within the β-subunit include protection by ATP/ADP of the E. colienzyme from proteolysis at Arg-80 (28Nishimura J.S. Ybarra J. Mann C.J. Mitchell T. J. Biol. Chem. 1993; 268: 13717-13722Abstract Full Text PDF PubMed Google Scholar). Furthermore, Steiner and Smith (11Steiner A.W. Smith R.A. J. Neurochem. 1981; 37: 582-593Crossref PubMed Scopus (19) Google Scholar) showed that ATP is more effective than GTP in labeling the α-subunit in brain, whereas only GTP is effective in liver. The 32-kDa phosphopeptide (α-subunit) thus labeled was the same as judged by two-dimensional electrophoresis. This is consistent with our conclusion that the β-subunit determines the specificity of SCS, although the α-subunit may contribute to the binding site. Studies of the nucleotide specificity of A-SCS and G-SCS in pigeon showed that each enzyme is highly specific for its nucleotide. Given that each form predominates in a tissue with a highly active Krebs cycle, it seems apparent that either form can fulfill the role of succinyl-CoA synthetase within the Krebs cycle. Our work shows that both forms are found in some tissues, although it remains to be determined if both are present within the same cell and the same mitochondrion. Jenkins and Weitzman (12Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1986; 205: 215-218Crossref PubMed Scopus (22) Google Scholar, 13Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1988; 230: 6-8Crossref PubMed Scopus (12) Google Scholar) have proposed that A-SCS participates in the Krebs cycle, whereas G-SCS synthesizes succinyl-CoA for heme synthesis and ketone body activation. The possible advantages of producing GTP rather than ATP within the Krebs cycle will be discussed in the accompanying paper (18Johnson J.D. Mehus J.G. Tews K. Milavetz B.I. Lambeth D.O. J. Biol. Chem. 1998; 273: 27580-27586Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Major differences in the apparent Michaelis constants were found for the two enzymes. The apparent K m for GDP is more than an order of magnitude less than that for ADP, and G-SCS is much more susceptible to substrate inhibition by GDP than A-SCS is by ADP. The physiological significance of this substrate inhibition, which would be of relevance to the direction of catalysis in the Krebs cycle, is uncertain. However, it seems reasonable that G-SCS is adapted to working at concentrations of GDP that are lower than those of ADP since the total concentration of guanine nucleotides in the matrix is about one-tenth that of adenine nucleotides, and the ratio of GTP to GDP in the mitochondrial matrix can be driven to a very high level (reviewed in Ref. 2Ottaway J.H. McClellan J.A. Saunderson C.L. Int. J. Biochem. 1981; 13: 401-410Crossref PubMed Scopus (44) Google Scholar). An evolutionary pressure to reduce or eliminate substrate inhibition by GDP may not have occurred because inhibition happens outside the physiological range. A similar argument has been advanced for nucleoside-diphosphate kinase (29Garces E. Cleland W.W. Biochemistry. 1969; 8: 633-640Crossref PubMed Scopus (106) Google Scholar). In the direction of succinyl-CoA synthesis, the K mof A-SCS for succinate is ∼10-fold that of G-SCS. In consideration of this fact, it is of interest to note that Jenkins and Weitzman (12Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1986; 205: 215-218Crossref PubMed Scopus (22) Google Scholar, 13Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1988; 230: 6-8Crossref PubMed Scopus (12) Google Scholar) have proposed that the role of G-SCS is to synthesize succinyl-CoA for heme biosynthesis and/or ketone body activation. In the accompanying paper (18Johnson J.D. Mehus J.G. Tews K. Milavetz B.I. Lambeth D.O. J. Biol. Chem. 1998; 273: 27580-27586Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), we present the sequences of the α-subunit and both β-subunits in pigeon. Demonstration that homologues of both β-sequences are expressed in several mammalian species supports enzymatic data suggesting that both A-SCS and G-SCS activities are found in many mammalian tissues. We thank W. S. Lane, R. Robinson, J. Neveu, and D. Arnelle (Harvard Microchemistry Facility) for preparation of the tryptic peptide maps. We also thank John Phipps (Macromolecular Resources, Colorado State University) for expertise in mass spectrometry." @default.
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