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Query: EC:1.1.1.41 (isocitrate dehydrogenase)
 3,101 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pig heart NADP-dependent isocitrate dehydrogenase is 65% inactivated by 3-bromo-2-ketoglutarate (Ehrlich, R.S., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,614-12,619) and 90% inactivated by 2-(4-bromo-2,3-dioxobutylthio)-1,N6- ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) (Bailey, J.M., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,620-12,626). Both inactivation reactions result in enzyme with an incorporation of 1.0 mol reagent/mol enzyme dimer and both modified enzymes bind only 1.0 mol manganous isocitrate or NADPH/mol enzyme dimer as compared to 2.0 mol manganous isocitrate or NADPH/mol enzyme dimer for unmodified enzyme. The inactivation reactions, which occur at or near the nucleotide binding site, are mutually exclusive. Reaction with either affinity reagent led to the isolation of the same modified triskaidekapeptide, DLAGXIHGLSNVK. We have isolated from isocitrate dehydrogenase a peptide, DLAGCIHGLSNVK, that had been modified by N-ethylmaleimide (NEM) with no loss of enzymatic activity. We now show that enzyme modified by NEM in the presence of isocitrate plus Mn2+ retains full catalytic activity but is not inactivated by either of the affinity reagents; thus, all three reagents appear to react at the same site. The analysis of HPLC tryptic maps of isocitrate dehydrogenase treated under denaturing conditions with iodo[3H]acetic acid or [3H]NEM demonstrates that both bromoketoglutarate and 2-BDB-T epsilon A-2',5'-DP react with the cysteine residue of DLAGCIHGLSNVK. We conclude that the cysteine of this triskaidekapeptide is close to the coenzyme binding site but is not essential for catalytic function.
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PMID:Inactivation of pig heart NADP-specific isocitrate dehydrogenase by two affinity reagents is due to reaction with a cysteine not essential for function. 153 72

The nucleotide affinity label 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP) reacts covalently with pig heart NAD+-dependent isocitrate dehydrogenase with a limiting value of 75% inactivation and loss of ADP activation concomitant with incorporation of about 1 mol of reagent/mol of average enzyme subunit (Huang, Y.-C., Bailey, J. M., and Colman, R. F. (1986) J. Biol. Chem. 251, 14100-14107). Complete protection against the functional changes is provided by ADP + Mn2+, and reagent incorporation is decreased to about 0.5 mol/mol of average enzyme subunit. We have now identified the critical modified peptide by comparison of the peptides labeled by 2-BDB-TADP at pH 6.8 in the absence and presence of ADP + Mn2+. After removal of excess reagent, modified enzyme was treated with [3H]NaBH4 to reduce the keto groups of the reagent and introduce a radioactive tracer into the reagent which is covalently linked to the protein. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated using two successive high performance liquid chromatography steps: 1) 0.1% trifluoroacetic acid with an acetonitrile gradient; and 2) 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas phase sequencing gave the modified peptide Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln in which aspartic acid is the target of 2-BDB-TADP. Isolation of the corresponding tryptic peptide from unmodified enzyme yielded the sequence Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln-CmCys-CmCys-Lys. Isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma), separable by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The evidence indicates that the specific peptide labeled by 2-BDB-TADP, which is at or near the ADP site, can be derived from the gamma subunit.
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PMID:Aspartyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate in the allosteric ADP site of pig heart NAD+-dependent isocitrate dehydrogenase. 274 37

2-(4-Bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) is an affinity label for the coenzyme-binding site of pig heart NADP+-dependent isocitrate dehydrogenase. Specific reaction occurs at the coenzyme site with an incorporation of 0.5 mol of reagent/mol of enzyme subunit (i.e. modification of only one subunit of the dimeric enzyme) (Bailey, J.M., and Colman, R.F. (1985) Biochemistry 24, 5367-5377). Modified enzyme, prepared by incubating 1 mg/ml isocitrate dehydrogenase with 75 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of substrate or coenzyme, was reduced with NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on DEAE-cellulose, followed by treatment with acid phosphatase (to decrease the negative charge by removing the phosphate groups from covalently bound reagent) and rechromatography on the same DEAE-cellulose column. The isolated peptides were characterized by amino acid analysis, dansylation, and gas-phase sequencing. A single triskaidekapeptide corresponding to modification of the coenzyme site by 2-BDB-T epsilon A-2',5'-DP was identified as: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Additional evidence indicated that X is a glutamate residue derivatized by 2-BDB-T epsilon A-2',5'-DP.
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PMID:Isolation of the glutamyl peptide labeled by the nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the active site of NADP+-specific isocitrate dehydrogenase. 288 70

Two new reactive adenine nucleotide analogues have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate (2-BDB-TA-2',5'-DP) and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2',5'-bisphosphate (2-BOP-TA-2',5'-DP). Starting with NADP+, 2'-phospho-adenosine 5'-(diphosphoribose) (PADPR) was generated enzymatically and was converted to PADPR 1-oxide by reaction with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 2',5'-bisphosphate (TA-2',5'-DP). Condensation of TA-2',5'-DP with 1,4-dibromobutanedione or 1,3-dibromo-2-propanone gave the final products 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP, respectively. The structure of these new reagents was determined by UV, 1H NMR, 31P NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Both of these reagents exhibit properties expected for an affinity label of the coenzyme site of NADP+-dependent isocitrate dehydrogenase. With both reagents, biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 6-7% residual activity, followed by a slower phase leading to total inactivation. The inactivation rate constants for both reagents exhibit a nonlinear dependence on reagent concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. The enzyme incorporates both reagents to a limited extent and is protected against inactivation by NADP+ and NADPH. The reaction of these new nucleotide analogues with isocitrate dehydrogenase is compared to the much slower inactivation caused by bromoacetone, indicating the importance of the nucleotide moiety in the functioning of the affinity labels. It is likely that 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP will have general applicability as affinity labels for other NADP+ binding enzymes.
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PMID:2-[(4-Bromo-2,3-dioxobutyl)thio]- and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2'5'-bisphosphate: new nucleotide analogues that act as affinity labels of nicotinamide adenine dinucleotide phosphate specific isocitrate dehydrogenase. 342 47

When the substrate isocitrate-Mn2+ is present, the fluorescent nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with pig heart NADP+-specific isocitrate dehydrogenase at the coenzyme binding site on one subunit of the dimeric enzyme [Bailey, J. M., & Colman, R. F. (1985) Biochemistry 24, 5367-5377]. The modified enzyme, which retains partial activity, binds 1 mol of NADPH or 1 mol of the coenzyme analogue, reduced thionicotinamide adenine dinucleotide phosphate (TNADPH), per dimer. TNADPH quenches the fluorescence of enzyme-bound 2-BDB-T epsilon A-2',5'-DP with an efficiency of energy transfer of 9.8%. From this value and the spectral properties of the donor and acceptor chromophores, a distance of 32 A was calculated as the average distance between coenzyme sites on the two subunits. Isocitrate dehydrogenase activity requires a divalent metal ion, such as Mn2+, Co2+, or Ni2+. Co2+ and Ni2+ have absorption spectra that overlap the emission spectra of enzyme-bound 2-BDB-T epsilon A-2',5'-DP. In the presence of isocitrate, each of these two metal ions quenches the fluorescence of the enzyme-bound reagent with an efficiency of energy transfer of 28-29%. From this value and the spectral characteristics of the energy donor and acceptors, an average distance of 8.0 A was estimated between the metal-isocitrate site and the labeled coenzyme site. These distances have provided constraints in formulating a model of the spatial arrangement of active-site ligands on isocitrate dehydrogenase.
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PMID:Distances among coenzyme and metal sites of NADP+-dependent isocitrate dehydrogenase using resonance energy transfer. 366 31

A new reactive ADP analogue has been synthesized: 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP). Reaction of ADP with m-chloroperoxybenzoic acid gave ADP 1-oxide, which was treated with NaOH, followed by reaction with carbon disulfide to yield 2-thioadenosine 5'-diphosphate. The final product was synthesized by condensation of 2-thioadenosine 5'-diphosphate with 1,4-dibromobutanedione. Reaction of pig heart NAD-specific isocitrate dehydrogenase with this nucleotide analogue (0.4 mM) causes a time-dependent loss of activity to a limiting value of 75% inactivation. The rate constant for inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TADP, with kmax = 0.021 min-1 and KI = 0.067 mM. Complete protection against inactivation by 0.2 mM 2-BDB-TADP is provided by ADP + Mn2+, but not by Mn2+ alone, isocitrate, alpha-ketoglutarate, or NAD. Incorporation of 2-BDB-TADP is proportional to the extent of inactivation, reaching 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. However, when inactivation is totally prevented by incubation with 2-BDB-TADP in the presence of ADP and Mn2+, 0.5 mol of reagent/mol of subunit is still incorporated, suggesting that inactivation may be attributed to 0.5 mol of reagent/mol of average subunit. In the native enzyme, the Km for total isocitrate is 1.8 mM and is decreased 6-fold to 0.3 mM in the presence of 1 mM ADP, whereas in the modified enzyme, with 25% residual activity, the Km for total isocitrate is about the same in the absence (2.0 mM) or presence (1.8 mM) of ADP. These results indicate that 2-BDB-TADP acts as an affinity label of the ADP allosteric site of NAD-dependent isocitrate dehydrogenase.
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PMID:Inactivation of NAD-dependent isocitrate dehydrogenase by affinity labeling of the allosteric ADP site by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate. 377 26

A new reactive fluorescent adenine nucleotide analogue has been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (BDB-T epsilon ADP). This compound reacts irreversibly with NADP+-specific isocitrate dehydrogenase. Biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 8-10% residual activity, followed by a slower phase leading to total inactivation. NADPH protects completely against the fast phase of the reaction, indicating that modification occurs at the coenzyme binding site, whereas isocitrate with MnSO4 protects totally against the slow phase of reaction. The inactivation rate constants for both phases exhibit nonlinear dependence on BDB-T epsilon ADP concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. Covalent incorporation of BDB-T epsilon ADP is limited and specific; only 0.99 mol of reagent/mol of subunit is incorporated when the enzyme is 98% inactivated in the absence of ligands. A maximum incorporation of 0.5 mol of reagent/mol of subunit is obtained in the presence of isocitrate and MnSO4, while incorporation in the presence of NADPH equals the difference between the incorporation in the absence of ligands and that measured in the presence of isocitrate and MnSO4. It appears that 0.5 mol of reagent/mol of subunit is responsible for the fast phase of inactivation and the remaining incorporation causes the slow phase. Under all conditions used in this study, isocitrate dehydrogenase has been shown to exist as a dimer by analytical ultracentrifugation and by cross-linking with dimethyl suberimidate followed by analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate. It is proposed that, in the fast phase of inactivation, 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate reacts at the coenzyme binding site of one subunit of dimeric isocitrate dehydrogenase, causing complete inactivation of the modified subunit and substantial inactivation of the other subunit. This new reagent is likely to have general applicability as an affinity label for other NADP+ binding enzymes.
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PMID:Affinity labeling of NADP+-specific isocitrate dehydrogenase by a new fluorescent nucleotide analogue, 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate. 407 1

Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP which reduces the Km of isocitrate. The new ADP analogue 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate (BDB-TADP) reacts irreversibly with the enzyme at pH 6.1 and 25 degrees C, causing a rapid loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and a slower inactivation measured using saturating isocitrate concentrations. The rate constant for loss of ADP activation exhibits a nonlinear dependence on BDB-TADP concentration; in the presence of 0.2 mM MnSO4, KI for the reversible enzyme-reagent complex is 0.069 mM with kmax at saturating reagent concentrations equal to 0.031 min-1. For reaction at the site causing overall inactivation, KI for the initial reversible enzyme-reagent complex is estimated to be 0.018 mM with kmax = 0.0083 min-1 in the presence of 0.2 mM MnSO4. Total protection against both reactions is provided by 1 mM ADP plus 0.2 mM MnSO4 or by 0.1 mM ADP plus 0.2 mM MnSO4 plus 0.2 mM isocitrate, but not by NAD, ATP, or ADP plus EDTA. The BDB-TADP thus appears to modify two distinct metal-dependent ADP-binding sites. Incubation of isocitrate dehydrogenase with 0.14 mM BDB-[beta-32P]TADP at pH 6.1 in the presence of 0.2 mM MnSO4 results in incorporation of 0.81 mol of reagent/mol of average subunit when the ADP activation is completely lost and the enzyme is 68% inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 0.5 mol of BDB-TADP/mol of average enzyme subunit causes complete loss of ADP activation, while reaction with another 0.5 mol of BDB-TADP would lead to total inactivation. The enzyme is composed of three distinct subunits in the approximate ratio 2 alpha:1 beta:1 gamma. The distribution of BDB-[beta-32P]TADP incorporated into modified enzyme is 63:30:7% for alpha:beta:gamma throughout the course of the reaction. These results indicate the 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate functions as an affinity label of two types of potential metal-dependent ADP sites of NAD-dependent isocitrate dehydrogenase and that these allosteric sites are present on two (alpha and beta) of the enzyme's three types of subunits.
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PMID:Affinity labeling of the allosteric ADP activation site of NAD-dependent isocitrate dehydrogenase by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate. 654 73

8-(4-Bromo-2,3-dioxobutylthio)nicotinamide adenine dinucleotide (8-BDB-TNAD), a new reactive NAD analog, was synthesized by coupling 8-thio-AMP with NMN, followed by condensation with 1,4-dibromobutanedione. Incubation of 160 microM 8-BDB-TNAD with the allosteric pig heart NAD-dependent isocitrate dehydrogenase causes time-dependent inactivation to a limit of 25% residual activity concomitant with incorporation of approximately 1 mol reagent/mol average subunit. In addition to binding sites for NAD and NADH, this enzyme has been shown to have regulatory sites for NADPH and for ADP (R. S. Ehrlich and R. F. Colman 1982, J. Biol. Chem. 257, 4769-4774). Marked protection against enzyme inactivation by 8-BDB-TNAD and incorporation is provided by the regulatory nucleotides NADPH or ADP, while NAD and NADH are less effective. The rate constant for inactivation shows a nonlinear dependence on 8-BDB-TNAD concentration which can be ascribed to reversible formation of an enzyme-reagent complex (KI = 83 microM) prior to an irreversible reaction (kmax = 0.0625 min-1). Analysis of the kinetic properties and binding characteristics of modified enzyme indicates that this enzyme retains the ability to bind ADP, but does not bind NADPH. Thus, 8-BDB-TNAD reacts at or near the allosteric NADPH site of pig heart NAD-dependent isocitrate dehydrogenase.
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PMID:8-(4-Bromo-2,3-dioxobutylthio)NAD: a new affinity label for NAD-specific isocitrate dehydrogenase. 810 65

Pig heart NAD-dependent isocitrate dehydrogenase reacts with 8-(4-bromo-2,3-dioxobutylthio)-NAD (8-BDB-TNAD) with incorporation of 1.21 mol of reagent/mol of average subunit when the enzyme reaches the limit of 25% residual activity (Kumar, A., and Colman, R. F., Arch. Biochem. Biophys. 308, 357-366, 1994). Inclusion of NADPH decreases both the extent of inactivation and the reagent incorporation to 0.55 mol/mol of average subunit. We have now isolated the peptides labeled by radioactive 8-(4-bromo-2,3-dioxobutylthio)-[2-3H]NAD and have located them within the sequence of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is composed of three types of subunits, present as alpha 2 beta gamma. We have separated the subunits from unmodified and 8-BDBT[2-3H]NAD-modified enzymes by HPLC on a C4 reverse-phase column, after pretreatment of the enzymes with sodium dodecyl sulfate or urea, and compared the subunit sequences of the porcine enzyme with those of the corresponding subunits from other mammalian NAD-dependent isocitrate dehydrogenases. The predominant radioactivity of 8-BDBT[2-3H]NAD is observed in the alpha and gamma peaks, and the NADPH-protected enzyme exhibits marked reduction in incorporation into these peaks. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the alpha-subunit is responsible for inactivation by 8-BDB-TNAD. Cyanogen bromide was used to cleave the modified enzyme, and we purified one labeled peptide from the alpha-subunit (amino acids 84-177) as well as one from the gamma-subunit (amino acids 67-186). In the alpha-subunit, decreased modification by [7-14C]-phenylglyoxal of Arg88 and Arg98 after prior labeling of the enzyme by 8-BDB-TNAD indicates that these residues are the critical target sites of the reactive nucleotide analogue. We conclude that alpha subunit's Arg88 and Arg98 are both at or near the allosteric NADPH sites of the pig heart isocitrate dehydrogenase.
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PMID:Identification of the subunits and target peptides of pig heart NAD-specific isocitrate dehydrogenase modified by the affinity label 8-(4-bromo-2,3-dioxobutylthio)NAD. 939 Jan 93


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