FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2020111263> ?p ?o ?g. }

• W2020111263 endingPage "7573" @default.
• W2020111263 startingPage "7567" @default.
• W2020111263 abstract "In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P. In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P. In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) 1EcIDHisocitrate dehydrogenase from E. coliBsIDHisocitrate dehydrogenase from B. subtilisIDH-K/Pisocitrate dehydrogenase kinase/phosphataseMOPS3-(N-morpholino)propanesulfonic acidHPLChigh performance liquid chromatographyMS/MStandem mass spectrometryLC/ESI/MS/MSliquid chromatography interfaced with an electrospray mass spectrometer operating in tandem mode for collision-induced dissociation is regulated by reversible phosphorylation (1Bennett P.M. Holms W.H. J. Gen. Microbiol. 1975; 87: 37-51Crossref PubMed Scopus (58) Google Scholar, 2Garnak M. Reeves H.C. Science. 1979; 203: 1111-1112Crossref PubMed Scopus (170) Google Scholar). Phosphorylation of EcIDH meters the flux of isocitrate between the Krebs cycle and the glyoxylate bypass, an anaplerotic pathway that is essential for growth on carbon sources such as acetate, ethanol, and fatty acids (3Kornberg H.L. Biochem. J. 1966; 99: 1-11Crossref PubMed Scopus (359) Google Scholar). isocitrate dehydrogenase from E. coli isocitrate dehydrogenase from B. subtilis isocitrate dehydrogenase kinase/phosphatase 3-(N-morpholino)propanesulfonic acid high performance liquid chromatography tandem mass spectrometry liquid chromatography interfaced with an electrospray mass spectrometer operating in tandem mode for collision-induced dissociation EcIDH was the first protein found to be regulated by phosphorylation in normal E. coli cells (2Garnak M. Reeves H.C. Science. 1979; 203: 1111-1112Crossref PubMed Scopus (170) Google Scholar), and it remains the most thoroughly investigated. Crystallographic studies revealed that the phosphorylation site, Ser-113, is buried within the active site crevice (4Hurley J.H. Thorsness P.E. Ramalingham V. Helmers N.H. Koshland D.E., Jr. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8635-8639Crossref PubMed Scopus (208) Google Scholar, 5Hurley J.H. Dean A.M. Thorsness P.E. Koshland D.E., Jr. Stroud R.M. J. Biol. Chem. 1990; 265: 3599-3602Abstract Full Text PDF PubMed Google Scholar). In the active, dephosphorylated state, the γ-hydroxyl of this serine hydrogen-bonds to the γ-carboxylate ofd-isocitrate (6Hurley J.H. Dean A.M. Sohl J.L. Koshland D.E., Jr. Stroud R.M. Science. 1990; 249: 1012-1016Crossref PubMed Scopus (210) Google Scholar, 7Hurley J.H. Dean A.M. Koshland D.E., Jr. Stroud R.M. Biochemistry. 1991; 30: 8671-8678Crossref PubMed Scopus (243) Google Scholar, 8Stoddard B.L. Dean A.M. Koshland D.E., Jr. Biochemistry. 1993; 32: 9310-9316Crossref PubMed Scopus (113) Google Scholar). Phosphorylation of Ser-113 inactivates EcIDH because the phosphate moiety prevents the formation of this hydrogen bond and provides a direct source of electrostatic repulsion (6Hurley J.H. Dean A.M. Sohl J.L. Koshland D.E., Jr. Stroud R.M. Science. 1990; 249: 1012-1016Crossref PubMed Scopus (210) Google Scholar, 9Thorsness P.E. Koshland D.E., Jr. J. Biol. Chem. 1987; 262: 10422-10425Abstract Full Text PDF PubMed Google Scholar, 10Dean A.M. Lee M.H.I. Koshland D.E., Jr. J. Biol. Chem. 1989; 264: 20482-20486Abstract Full Text PDF PubMed Google Scholar) and steric hindrance (11Dean A.M. Koshland D.E., Jr. Science. 1990; 249: 1044-1046Crossref PubMed Scopus (101) Google Scholar) with the γ-carboxylate of isocitrate. The IDH phosphorylation cycle is catalyzed by IDH kinase/phosphatase (IDH-K/P), a bifunctional enzyme encoded by aceK (12LaPorte D.C. Koshland D.E., Jr. Nature. 1982; 300: 458-460Crossref PubMed Scopus (148) Google Scholar, 13LaPorte D.C. Thorsness P.E. Koshland D.E., Jr. J. Biol. Chem. 1985; 260: 10563-10568Abstract Full Text PDF PubMed Google Scholar). The phosphatase activity has an absolute requirement for ATP or ADP (12LaPorte D.C. Koshland D.E., Jr. Nature. 1982; 300: 458-460Crossref PubMed Scopus (148) Google Scholar). In addition, this protein has an intrinsic ATPase activity (14Stueland C.S. Eck K.R. Stieglbauer K.T. LaPorte D.C. J. Biol. Chem. 1987; 262: 16095-16099Abstract Full Text PDF PubMed Google Scholar). The results of affinity labeling and analyses of the effects of site-directed and random mutagenesis of aceK suggest that all three activities are located in the same active site (15Miller S.P. Karschnia E.J. Ikeda T.P. LaPorte D.C. J. Biol. Chem. 1996; 271: 19124-19128Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 16LaPorte D.C. J. Cell. Biochem. 1993; 51: 14-18Crossref PubMed Scopus (71) Google Scholar, 17Stueland C.S. Ikeda C.S. LaPorte D.C. J. Biol. Chem. 1989; 264: 13775-13779Abstract Full Text PDF PubMed Google Scholar, 18Varela I. Nimmo H.G. FEBS Lett. 1988; 231: 361-365Crossref PubMed Scopus (11) Google Scholar). Our working model proposes that the phosphatase reaction results from the kinase back reaction tightly coupled to ATP hydrolysis (15Miller S.P. Karschnia E.J. Ikeda T.P. LaPorte D.C. J. Biol. Chem. 1996; 271: 19124-19128Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 16LaPorte D.C. J. Cell. Biochem. 1993; 51: 14-18Crossref PubMed Scopus (71) Google Scholar). One of our goals is to understand the structural basis for the recognition of EcIDH by IDH-K/P. As an approach to this problem, we have employed the isocitrate dehydrogenase from Bacillus subtilis (BsIDH) as a substrate analogue for IDH-K/P. Although BsIDH does not appear to be phosphorylated in vivo, 2A. L. Sonenshein, personal communication. it maintains extensive sequence and structural similarity with EcIDH (19Singh S.K. Matsuno K. LaPorte D.C. Banaszak L.J. J. Biol. Chem. 2001; 276: 26154-26163Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The structural congruence includes the active site, where almost all of the residues implicated in ligand binding and catalysis are conserved (19Singh S.K. Matsuno K. LaPorte D.C. Banaszak L.J. J. Biol. Chem. 2001; 276: 26154-26163Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Here, we extend the comparative analysis between BsIDH and EcIDH to include the dehydrogenase reaction, along with a quantitative examination of the effect of phosphorylation on IDH activity. We also demonstrate that BsIDH is phosphorylated once per monomer and on the corresponding active site serine. Despite the similarities revealed by these experiments, BsIDH is a strikingly poor substrate of IDH-K/P. These data are interpreted in light of our recently solved crystal structure of BsIDH (19Singh S.K. Matsuno K. LaPorte D.C. Banaszak L.J. J. Biol. Chem. 2001; 276: 26154-26163Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). EcIDH was overexpressed from the multicopy plasmid pSML1, atet R derivative of pTK509 (13LaPorte D.C. Thorsness P.E. Koshland D.E., Jr. J. Biol. Chem. 1985; 260: 10563-10568Abstract Full Text PDF PubMed Google Scholar), that had been transformed into E. coli strain ST2010R (15Miller S.P. Karschnia E.J. Ikeda T.P. LaPorte D.C. J. Biol. Chem. 1996; 271: 19124-19128Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). BsIDH was overexpressed from plasmid pKM14 that had been transformed into theE. coli icd-deletion strain KME44 (19Singh S.K. Matsuno K. LaPorte D.C. Banaszak L.J. J. Biol. Chem. 2001; 276: 26154-26163Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). EcIDH was purified by a modification of the method employed in Garnak and Reeves (20Garnak M. Reeves H.C. J. Biol. Chem. 1979; 254: 7915-7920Abstract Full Text PDF PubMed Google Scholar). Specifically, cells were disrupted in a French press (12,600 psi) instead of a sonicator, and a gel filtration column (Sephacryl S-300 High Resolution, 2.5 × 130 cm, Amersham Biosciences, Inc.) was used as the final step instead of Affi-Gel Blue (Bio-Rad) chromatography. BsIDH was purified in a manner similar to that employed for EcIDH except that the initial protamine sulfate and ammonium sulfate steps were omitted. Instead, lysed cells were centrifuged at 46,000 ×g for 90 min at 4 °C to remove debris and directly applied to a pre-equilibrated DEAE-Sepharose column (2.5 × 48 cm, Amersham Biosciences, Inc.). IDH-K/P was overexpressed in E. coli strain ST2010R harboring the plasmid pEKL1 as described previously (15Miller S.P. Karschnia E.J. Ikeda T.P. LaPorte D.C. J. Biol. Chem. 1996; 271: 19124-19128Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). It was then purified via DEAE-Sepharose and IDH-Sepharose chromatographies as published elsewhere (12LaPorte D.C. Koshland D.E., Jr. Nature. 1982; 300: 458-460Crossref PubMed Scopus (148) Google Scholar). Both phospho-EcIDH and phospho-BsIDH were prepared in vitrousing purified IDH-K/P and ATP. Phospho-IDHs were then separated from IDH-K/P, dephospho-IDH, Pi, and any remaining nucleotides by applying the reaction mixture to a column composed of a top layer of Bio-Gel P6 resin (Bio-Rad) and a bottom layer of Affi-Gel Blue resin, as outlined previously (21LaPorte D.C. Koshland D.E., Jr. Nature. 1983; 305: 286-290Crossref PubMed Scopus (124) Google Scholar). All proteins were greater than 95% pure as judged by SDS-polyacrylamide gel electrophoresis (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) followed by Coomassie Blue staining (data not shown). Protein concentrations were determined by the Peterson protocol (23Peterson G.L. Anal. Biochem. 1979; 100: 201-220Crossref PubMed Scopus (886) Google Scholar), a variation of the Lowry method (24Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), using bovine serum albumin as the standard. EcIDH and BsIDH activities were measured by monitoring the reduction of NADP+ to NADPH at 37 °C in an ISS K-2 spectrofluorometer with excitation and emission wavelengths of 350 and 459 nm, respectively. The standard reaction (500 μl) contained 25 mm MOPS (pH 7.5), 100 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 4 μg/ml bovine serum albumin, and variable concentrations of NADP+ andd-isocitrate (prepared with equimolar MgCl2). Reactions were initiated by the addition of 8 ng of either EcIDH or BsIDH, and the increase in fluorescence intensity (F.I.) was followed over the course of 1.5 min. Initial rates were determined via linear regression of the change in F.I. with time. The correlation between intensity and micromolars of NADPH was determined by measuring the F.I. for a known amount of NADPH generated by the complete conversion of NADP+ in the presence of excessd-isocitrate and IDH. NADP+ concentrations were ascertained spectrophotometrically by measuring A 259 with a molar extinction coefficient of 18,000 m−1cm−1 and a path length of 1 cm. d-isocitrate concentrations were determined by completely converting isocitrate to α-ketoglutarate in the presence of excess NADP+ and then by measuring the amount of NADPH produced. The resulting NADPH concentration is equimolar with α-ketoglutarate and was determined by measuring A 340 with a molar extinction coefficient of 6,220 m−1 cm−1 and a path length of 1 cm. For calculation of kinetic constants, the concentration ofd-isocitrate was varied at several fixed concentrations of NADP+. The average initial velocity was obtained from at least three measurements for each pair of substrate concentration. The data were graphed as Lineweaver-Burk plots, and linear regression was then employed to construct slope and intercept replots (25Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, New York1975Google Scholar). These replots were linear and were subsequently used to calculate the values of the kinetic parameters. The stoichiometry of phosphorylation and the effect on IDH activity were ascertained by simultaneously quantitating the incorporation of [32P]phosphate into IDH and by measuring the resulting decrease in IDH activity, as described previously (21LaPorte D.C. Koshland D.E., Jr. Nature. 1983; 305: 286-290Crossref PubMed Scopus (124) Google Scholar). The standard reaction (350 μl) contained 25 mm MOPS (pH 7.5), 100 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.5 mm EDTA, 0.4 mg/ml bovine serum albumin, 100 μm [γ-32P]ATP (∼300 cpm/pmol, prepared with equimolar MgCl2), and 2.0 μm of either EcIDH or BsIDH. Duplicate reactions for each IDH were initiated by adding IDH-K/P (441 ng for EcIDH or 2,674 ng for BsIDH) and incubated at 37 °C. At every time point indicated in Fig. 1, a 50-μl aliquot of each reaction was removed, terminated with 10 μl of 120 mmEDTA, and placed on ice. The production of [32P]phospho-IDH was monitored by precipitating the protein from 20 μl of each terminated sample onto filter paper with trichloroacetic acid and then measuring the amount of radioactivity following extensive washing of the filter paper squares, as outlined elsewhere (21LaPorte D.C. Koshland D.E., Jr. Nature. 1983; 305: 286-290Crossref PubMed Scopus (124) Google Scholar). IDH activity was assayed spectrophotometrically by monitoring the production of NADPH at A 340 at 37 °C. The dehydrogenase assay contained 25 mm MOPS (pH 7.5), 0.25 mm NADP+, 0.5 mmd,l-isocitrate, and 5 mmMgCl2. Five μg of purified dephospho-BsIDH or phospho-BsIDH were electrophoresed on a Bio-Rad Mini-Protean III system using 12.5% acrylamide gels of 1.5-mm thickness and then silver-stained with the Bio-Rad Silver Stain Plus kit. Protein bands were excised, destained, and digested with sequencing-grade modified trypsin (625 μg/band, Promega) according to a protocol adapted from Shevchenko et al. (26Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). Extracted, dehydrated peptides were subsequently reconstituted in 15 μl of 4% acetonitrile, 0.2% acetic acid just prior to mass spectrometric analysis. Tryptic peptides were separated on a reverse-phase capillary HPLC system (Magic 2002, Michrome BioResources) employing a C18 column (0.075 × 70 mm, 5-μm particle diameter, 200-Å pore size) with mobile phases of 2% acetonitrile, 0.2% acetic acid (solution A) and 97.8% acetonitrile, 0.2% acetic acid (solution B). The flow rate was set to run at 450 nl/min over 60 min. The HPLC was connected on-line to an LCQ Classic electrospray ion trap mass spectrometer (ThermoFinnigan) for direct measurement of peptides as they eluted from the column. The instrument was programmed to automatically switch to tandem mass spectrometry (MS/MS) mode and acquire collision-induced dissociation spectra if the intensity of the parent ion peaks exceeded a preset threshold. During the MS/MS phase, charged peptides isolated in the ion trap are bombarded with neutral argon, xenon, or helium molecules (27Siuzdak G. Mass Spectrometry for Biotechnology. Academic Press, San Diego, CA1996Google Scholar); helium gas was used in the LCQ Classic. This collision induces relatively mild fragmentation, with multiply charged species fragmenting more easily than singly charged ones (28DeGnore J.P. Qin J. J. Am. Soc. Mass Spectrom. 1998; 9: 1175-1188Crossref PubMed Scopus (235) Google Scholar). Fragmentation occurring along the peptide backbone generates two classes of daughter ions: b n ions are those produced by cleavage from the C terminus but which retain charge on the N terminus; y n ions are those produced by cleavage from the N terminus but which retain charge on the C terminus. Therefore, b-type ions include N-terminal amino acids, whereas y-type ions include C-terminal amino acids (27Siuzdak G. Mass Spectrometry for Biotechnology. Academic Press, San Diego, CA1996Google Scholar, 29Biemann K. Biomed. Environ. Mass Spectrom. 1988; 16: 99-111Crossref PubMed Scopus (925) Google Scholar). Phosphopeptides were initially identified by constructing a “data base” consisting of BsIDH and then searching this minimized data base with the program SEAQUEST (30Eng J.K. McCormack A.L. Yates III, J.R. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5472) Google Scholar, 31Yates III, J.R. Eng J.K. McCormack A.L. Schieltz D. Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1109) Google Scholar). The user interface permitted us to add a theoretical phosphate moiety to every serine, threonine, and tyrosine in the amino acid sequence. The program was then able to consider every peptide as both modified and unmodified, compare the theoretical tandem mass spectra with actual fragmentation patterns, and calculate a correlation coefficient. The MS/MS scans selected by SEAQUEST were then visualized with the Qual Browser software from ThermoFinnigan and manually scrutinized for the presence of the expected b n and y n daughter ions. IDH kinase activity was measured by monitoring the transfer of [32P]phosphate from [γ-32P]ATP to IDH at 37 °C, as described previously (21LaPorte D.C. Koshland D.E., Jr. Nature. 1983; 305: 286-290Crossref PubMed Scopus (124) Google Scholar). The standard reaction mixture (50 μl) contained 25 mm MOPS (pH 7.5), 100 mm NaCl, 5 mmMgCl2, 1 mm dithiothreitol, 0.5 mmEDTA, 0.2 mg/ml bovine serum albumin, 1 mm[γ-32P]ATP (saturating, ∼1 × 107cpm/assay, prepared with equimolar MgCl2) and variable concentrations of either EcIDH or BsIDH. The reactions, performed in triplicate for each concentration, were initiated by the addition of IDH-K/P (8.4 ng for EcIDH or 42 ng for BsIDH) and then incubated at 37 °C for 20 min. The production of [32P]phospho-IDH was quantitated by acid-precipitating the protein onto filter paper and then measuring the amount of radioactivity by liquid scintillation counting. IDH phosphatase activity was measured by monitoring the release of [32P]phosphate from [32P]phospho-IDH at 37 °C, as described previously (21LaPorte D.C. Koshland D.E., Jr. Nature. 1983; 305: 286-290Crossref PubMed Scopus (124) Google Scholar). The standard reaction (50 μl) contained 25 mm MOPS (pH 7.5), 100 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.5 mm EDTA, 2 mg/ml bovine serum albumin, 1 mm ATP (saturating, prepared with equimolar MgCl2) and variable concentrations of either [32P]phospho-EcIDH or [32P]phospho-BsIDH (∼5 × 104cpm/assay). The reactions, performed in triplicate for each concentration, were initiated by the addition of IDH-K/P (4.2 ng for phospho-EcIDH or 1,300 ng for phospho-BsIDH) and then incubated for 20 min. at 37 °C. The amount of [32P]phosphate released was quantitated in the supernatant following acid precipitation of the protein with trichloroacetic acid. Kinetic constants were obtained by linear regression of Lineweaver-Burk plots of the data. BsIDH is structurally similar to EcIDH, making this protein a good candidate for a substrate analogue of E. coliIDH-K/P. It is particularly noteworthy that almost all of the residues implicated in substrate binding and catalysis in the two IDHs are conserved and occupy virtually identical positions in three-dimensional space (19Singh S.K. Matsuno K. LaPorte D.C. Banaszak L.J. J. Biol. Chem. 2001; 276: 26154-26163Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This conservation includes Ser-113, a residue that plays a dual role as a contributor to isocitrate binding and as the site of phosphorylation in EcIDH. To further characterize the extent of conservation between EcIDH and BsIDH, we examined the initial velocity kinetics of their respective dehydrogenase activities. Measurements of the initial velocities of BsIDH and EcIDH both produced intersecting patterns on Lineweaver-Burk plots (data not shown), indicative of a sequential (ordered or random) mechanism. Both enzymes displayed nearly equivalent maximum velocities and Michaelis constants for d-isocitrate and NADP+ (Table I). The only significant difference between these proteins was the observation that the apparent dissociation and Michaelis constants for each substrate are nearly identical for EcIDH but not for BsIDH. However, the precise significance of this disparity is uncertain since EcIDH appears to act via a steady-state random bi ter mechanism (32Dean A.M. Koshland D.E., Jr. Biochemistry. 1993; 32: 9302-9309Crossref PubMed Scopus (68) Google Scholar), and such mechanisms are governed by extremely complex rate laws (25Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, New York1975Google Scholar). Thus, despite this minor difference, the kinetic behaviors of EcIDH and BsIDH are quite similar, as would be predicted from the extensive sequence and structural congruence between them.Table IKinetic constants for BsIDH and EcIDHEnzymeD-IsocitrateNADP+K m1-aKm = Michaelis constant. The values are given in μm concentrations.K1-bK = apparent dissociation constant. The values are given in μm concentrations.α = K/KmK m 1-aKm = Michaelis constant. The values are given in μm concentrations.K1-bK = apparent dissociation constant. The values are given in μm concentrations.α = K/KmV max1-cThe values are given in terms of nmol/min/μg enzyme.BsIDH5.9 ± 0.914.5 ± 1.62.5 ± 0.214.5 ± 2.236.2 ± 6.52.5 ± 0.2107 ± 7EcIDH4.9 ± 0.24.0 ± 0.50.8 ± 0.119.6 ± 3.714.6 ± 1.40.8 ± 0.1135 ± 7The data shown are the average ± S.E. obtained from at least three independent experiments.1-a Km = Michaelis constant. The values are given in μm concentrations.1-b K = apparent dissociation constant. The values are given in μm concentrations.1-c The values are given in terms of nmol/min/μg enzyme. Open table in a new tab The data shown are the average ± S.E. obtained from at least three independent experiments. The second step in our comparative analysis between BsIDH and EcIDH was to ascertain whether BsIDH could be phosphorylated in vitro by IDH-K/P. Preliminary experiments combining the purified proteins with [γ−32P]ATP and examining the reaction mixture via SDS-PAGE followed by autoradiography demonstrated that BsIDH could indeed could be phosphorylated (data not shown). We subsequently determined the stoichiometry of the phosphorylation reaction and its effect on dehydrogenase activity. This was accomplished with a time course experiment in which the incorporation of [32P]phosphate into either BsIDH or EcIDH was monitored along with any corresponding decrease in IDH activity. As shown in Fig. 1, stoichiometric phosphorylation resulted in essentially complete inhibition of dehydrogenase activity for both BsIDH as well as EcIDH. These data imply that, in each enzyme, there is only one phosphate group per subunit and that this phosphate is responsible for the loss of IDH activity. Mass spectrometry was employed to determine whether BsIDH was phosphorylated on Ser-104, the residue homologous to the phosphorylation site in EcIDH, Ser-113. Both dephospho-BsIDH and phospho-BsIDH were subjected to in-gel trypsin digestion, and the resulting peptides were analyzed via LC/ESI/MS/MS (liquid chromatography connected on-line to an electrospray ion trap mass spectrometer operating in tandem mode). Data for dephospho-BsIDH were examined first. The tryptic peptide containing the putative phosphorylation site, 104-SLNVALR-110, has a predicted unprotonated monoisotopic mass of 771.47 Da (m/z 772.47 and m/z 386.74 for the singly and doubly charged species, respectively). A peptide with these features eluted from the column at 21.83 min, and the MS/MS spectrum of the doubly charged ion is shown in Fig.2 A. As indicated on the figure and in Table II, five of seven theoretically possible b n ions (singly charged) and six of seven theoretically possible y n ions (singly charged) were observed. This unambiguously identifies the sequence of this peptide as SLNVALR.Table IISingly charged bn and yn ions present in MS/MS spectra of tryptic peptide 104-SLNVALR-110BsIDHPhospho-BsIDHSequenceNo.b n 2-ab n and y n are given in terms of m/z.SequenceNo.y n 2-ab n and y n are given in terms of m/z.SequenceNo.b n 2-ab n and y n are given in terms of m/z.b nΔ 2-bb nΔ refers to the corresponding b n ion minus H3PO4.SequenceNo.y n 2-ab n and y n are given in terms of m/z.S1SLNVALR7pS1pSLNVALR7SL2200.8LNVALR6685.3pSL2280.8182.8LNVALR6685.4SLN3314.8NVALR5572.3pSLN3395.5297.1NVALR5572.3SLNV4413.9VALR4458.2pSLNV4494.0396.1VALR4458.2SLNVA5485.0ALR3359.2pSLNVA5565.0466.9ALR3359.1SLNVAL6598.1LR2288.0pSLNVAL6677.7580.2LR2288.0SLNVALR7R1175.0pSLNVALR7R1174.92-a b n and y n are given in terms of m/z.2-b b nΔ refers to the corresponding b n ion minus H3PO4. Open table in a new tab With this result in hand, data for phospho-BsIDH were analyzed next. If the peptide contained a phosphate group, then its predicted unprotonated monoisotopic mass would be 851.43 Da (m/z 852.43 and m/z 426.72 for the singly and doubly charged species, respectively). A peptide with these features eluted from the HPLC column at 15.98 min, and the MS/MS fragmentation spectrum of the doubly charged species is shown in Fig. 2 B. As indicated on the figure and in Table II, five of seven theoretically possible b n ions (singly charged) and six of seven theoretically possible y n ions (singly charged) were present. Close inspection reveals that each of the b n ions was ∼80 Da (mass of the phosphate moiety) greater than the corresponding b n ions from the dephospho-peptide. This, along with the parent ion at m/z 426.7, verifies the sequence of this peptide as pSLNVALR. Further confirmation is provided by a second series of singly charged b n ions, denoted b nΔ (33Zhang X. Herring C.J. Romano P.R. Szczepanowska J. Brzeska H. Hinnebusch A.G. Qin J. Anal. Chem. 1998; 70: 2050-2059Crossref PubMed Scopus (182) Google Scholar), that were 98 Da (mass of H3PO4) less than the corresponding b n ions. It has been documented that in ion trap MS/MS, phosphoserine loses H3PO4 via β-elimination to produce dehydroalanine. This 98-Da loss is the unequivocal fingerprint of a phosphopeptide, particularly one containing a phosphoserine (28DeGnore J.P. Qin J. J. Am. Soc. Mass Spectrom. 1998; 9: 1175-1188Crossref PubMed Scopus (235) Google Scholar). The sequences and structures of EcIDH and BsIDH are extremely similar, as are their dehydrogenase kinetics. Furthermore, both proteins were phosphorylated by E. coli IDH-K/P once per monomer and on corresponding residues. We finally examined the kinetics of the IDH kinase and phosphatase reactions. Despite the extensive congruence between these proteins, the Michaelis constant" @default.
• W2020111263 created "2016-06-24" @default.
• W2020111263 creator A5011492323 @default.
• W2020111263 creator A5016726674 @default.
• W2020111263 creator A5025494009 @default.
• W2020111263 creator A5057013603 @default.
• W2020111263 creator A5087169660 @default.
• W2020111263 date "2002-03-01" @default.
• W2020111263 modified "2023-10-16" @default.
• W2020111263 title "Bacillus subtilis Isocitrate Dehydrogenase" @default.
• W2020111263 cites W136965677 @default.
• W2020111263 cites W1496388590 @default.
• W2020111263 cites W1537169897 @default.
• W2020111263 cites W1555898673 @default.
• W2020111263 cites W1569018409 @default.
• W2020111263 cites W1572613835 @default.
• W2020111263 cites W1592200831 @default.
• W2020111263 cites W1714732282 @default.
• W2020111263 cites W1775749144 @default.
• W2020111263 cites W1966463163 @default.
• W2020111263 cites W1976223036 @default.
• W2020111263 cites W2001769819 @default.
• W2020111263 cites W2002161747 @default.
• W2020111263 cites W2002609852 @default.
• W2020111263 cites W2003695026 @default.
• W2020111263 cites W2011039500 @default.
• W2020111263 cites W2017372126 @default.
• W2020111263 cites W2018948641 @default.
• W2020111263 cites W2020209954 @default.
• W2020111263 cites W2026465178 @default.
• W2020111263 cites W2028701325 @default.
• W2020111263 cites W2028961289 @default.
• W2020111263 cites W2033756350 @default.
• W2020111263 cites W2044973253 @default.
• W2020111263 cites W2051531989 @default.
• W2020111263 cites W2051719256 @default.
• W2020111263 cites W2059572899 @default.
• W2020111263 cites W2078218666 @default.
• W2020111263 cites W2078594804 @default.
• W2020111263 cites W2078977165 @default.
• W2020111263 cites W2079937312 @default.
• W2020111263 cites W2100837269 @default.
• W2020111263 cites W2117611385 @default.
• W2020111263 cites W2145908525 @default.
• W2020111263 cites W4246536726 @default.
• W2020111263 doi "https://doi.org/10.1074/jbc.m107908200" @default.
• W2020111263 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11751849" @default.
• W2020111263 hasPublicationYear "2002" @default.
• W2020111263 type Work @default.
• W2020111263 sameAs 2020111263 @default.
• W2020111263 citedByCount "31" @default.
• W2020111263 countsByYear W20201112632012 @default.
• W2020111263 countsByYear W20201112632013 @default.
• W2020111263 countsByYear W20201112632014 @default.
• W2020111263 countsByYear W20201112632017 @default.
• W2020111263 countsByYear W20201112632018 @default.
• W2020111263 countsByYear W20201112632019 @default.
• W2020111263 countsByYear W20201112632021 @default.
• W2020111263 crossrefType "journal-article" @default.
• W2020111263 hasAuthorship W2020111263A5011492323 @default.
• W2020111263 hasAuthorship W2020111263A5016726674 @default.
• W2020111263 hasAuthorship W2020111263A5025494009 @default.
• W2020111263 hasAuthorship W2020111263A5057013603 @default.
• W2020111263 hasAuthorship W2020111263A5087169660 @default.
• W2020111263 hasBestOaLocation W20201112631 @default.
• W2020111263 hasConcept C181199279 @default.
• W2020111263 hasConcept C185592680 @default.
• W2020111263 hasConcept C2777150147 @default.
• W2020111263 hasConcept C2777272437 @default.
• W2020111263 hasConcept C2781330172 @default.
• W2020111263 hasConcept C523546767 @default.
• W2020111263 hasConcept C54355233 @default.
• W2020111263 hasConcept C55493867 @default.
• W2020111263 hasConcept C86803240 @default.
• W2020111263 hasConcept C89423630 @default.
• W2020111263 hasConceptScore W2020111263C181199279 @default.
• W2020111263 hasConceptScore W2020111263C185592680 @default.
• W2020111263 hasConceptScore W2020111263C2777150147 @default.
• W2020111263 hasConceptScore W2020111263C2777272437 @default.
• W2020111263 hasConceptScore W2020111263C2781330172 @default.
• W2020111263 hasConceptScore W2020111263C523546767 @default.
• W2020111263 hasConceptScore W2020111263C54355233 @default.
• W2020111263 hasConceptScore W2020111263C55493867 @default.
• W2020111263 hasConceptScore W2020111263C86803240 @default.
• W2020111263 hasConceptScore W2020111263C89423630 @default.
• W2020111263 hasIssue "9" @default.
• W2020111263 hasLocation W20201112631 @default.
• W2020111263 hasOpenAccess W2020111263 @default.
• W2020111263 hasPrimaryLocation W20201112631 @default.
• W2020111263 hasRelatedWork W1548672655 @default.
• W2020111263 hasRelatedWork W2058770031 @default.
• W2020111263 hasRelatedWork W2120639575 @default.
• W2020111263 hasRelatedWork W2418244915 @default.
• W2020111263 hasRelatedWork W2511175281 @default.
• W2020111263 hasRelatedWork W2972415784 @default.
• W2020111263 hasRelatedWork W3010800646 @default.
• W2020111263 hasRelatedWork W3041516390 @default.
• W2020111263 hasRelatedWork W346701668 @default.

• next