3-Bromo-2-ketoglutarate: a substrate and affinity label for DPN-dependent isocitrate dehydrogenase

Bednar, Rodney A.; Hartman, Fred C.; Colman, Roberta F.

doi:10.1021/bi00258a025

Cited by 13 publications

(6 citation statements)

References 35 publications

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“…ADP has been shown to decrease the Km (Cohen & Colman, 1972) as well as the KO for isocitrate. The effector also protected the substrate binding site of isocitrate dehydrogenase from modification by 2,3-butanedione (Hayman & Colman, 1978) and 3-bromo-2-ketoglutarate (Bednar et al, 1982a). These previous results indicated conformational changes in the catalytic site upon binding of ADP to the allosteric site.…”

Section: Discussionmentioning

confidence: 99%

Affinity labeling of nicotinamide adenine dinucleotide-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate. Evidence for the formation of an unusual reaction product

King¹,

Colman²

1983

Biochemistry

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Modification of the pig heart NAD-dependent iswitrate dehydrogenase by the 2',3'-dialdehyde derivative of ADP (oADP) resulted in a time-dependent inactivation of the enzyme. Two kinetically distinct phases are observed for the loss in enzymatic activity with maximum rate constants of 0.38 and 0.023 min-', at saturating concentrations of oADP, at pH Abbreviations: oADP, 2',3'-dialdehyde derivative of ADP; Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid); oATP, 2',3'-dialdehyde derivative of ATP; ACS, aqueous counting scintillant; Tris, tris(hydroxy-methy1)aminomethane; EDTA, ethylenediaminetetraacetic acid.

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Section: Discussionmentioning

confidence: 99%

Affinity labeling of nicotinamide adenine dinucleotide-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate. Evidence for the formation of an unusual reaction product

King¹,

Colman²

1983

Biochemistry

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“…As the physiological substrate of the enzyme is Mg2+-isocitrate, it is reasonable to postulate two regulatory and two catalytic isocitrate binding sites per tetramer. Two affinity labels have been used to explore isocitrate binding to NAD+-ICDH: 3-bromo-2-oxoglutarate [29] and 3,4-didehydro-2-oxoglutarate [30]. Both are incorporated into all subunits, at a ratio of 1 mol label: 1 mol subunit.…”

Section: Comparison Of Mammalian Yeast and Bacterial Icdh Sequencesmentioning

confidence: 99%

Molecular cloning and deduced amino acid sequences of the α- and β- subunits of mammalian NAD+-isocitrate dehydrogenase

Nichols

Perry

Hall

et al. 1995

Biochemical Journal

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A 153 bp fragment of the cDNA encoding the beta-subunit of pig heart NAD(+)-isocitrate dehydrogenase (NAD(+)-ICDH) was specifically amplified by PCR, using redundant oligonucleotide primers based on partial peptide sequence data [Huang and Colman (1990) Biochemistry 29, 8266-8273]. This PCR fragment was then used as a probe to isolate cDNA clones encoding the complete mature form of the beta-subunit from a monkey testis cDNA library. Examination of the deduced amino acid sequence of the monkey subunit and the partial sequence of the pig heart enzyme revealed a high level of sequence conservation. In addition, 3 overlapping fragments of the cDNA for the alpha-subunit of monkey NAD(+)-ICDH were amplified using oligonucleotide primers derived from the cDNA sequence of a subunit of bovine NAD(+)-ICDH (EMBL accession no: U07980). These cDNA fragments allow deduction of the amino acid sequence of the alpha-subunit. Since the gamma-subunit of monkey NAD(+)-ICDH has already been cloned [Nichols, Hall, Perry and Denton (1993) Biochem. J. 295, 347-350], a deduced amino acid sequence is now available for all three subunits of mammalian NAD(+)-ICDH. Interrelationships between these subunits are discussed and they are compared with the two subunits of yeast NAD(+)-ICDH and Escherichia coli NADP(+)-ICDH.

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“…Although BrKG is a dicarboxylic acid, its electronegative bromine atom may occupy the site of the central carboxylate of the natural substrates of isocitrate dehydrogenase. The rate constant for inactivation exhibits a nonlinear dependence on BrKG concentration, consistent with reversible formation of an enzyme-reagent complex prior to irreversible reaction (Bednar et al, 1982a). Specific protection against inactivation is provided by isocitrate in the presence of Mn2+, consistent with the requirement of divalent metal ion for tight binding of the substrate to the enzyme .…”

Section: Separation By Hplc Of [Hc\{carboxymethyl)cysteinementioning

confidence: 62%

“…Pig heart NAD+-dependent isocitrate dehydrogenase was inactivated 80% by incubation for 150 min with 5 mM 3-bromo-2-ketoglutarate at pH 6.1. Enzyme reacted under similar conditions with 3-bromo-2-[14C]ketoglutarate has been shown to incorporate 0.7 mol of reagent/mol of average subunit (Bednar et al, 1982a). After unreacted BrKG was removed, the carbonyl group of enzyme-bound reagent was reduced with [3H]NaBH4, yielding enzyme labeled with tri-tium at the reacted amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…The peptide modified by 3-bromo-2-ketoglutarate, which is in or near the substrate site, is derived only from the separated y subunit. reaction with 3-bromo-2-ketoglutarate (BrKG),1 *which acts as an affinity label of the isocitrate binding site (Bednar et al, 1982a). A marked decrease in the rate constant for in- activation was produced by isocitrate in the presence of Mn24", whereas the coenzymes NAD4" and NADH were much less effective in preventing inactivation.…”

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Cysteinyl peptide labeled by 3-bromo-2-ketoglutarate in the active site of pig heart NAD+-dependent isocitrate dehydrogenase

Saha

Huang²,

Colman³

1989

Biochemistry

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The substrate affinity label 3-bromo-2-ketoglutarate (BrKG) reacts covalently with pig heart NAD+-specific isocitrate dehydrogenase with complete inactivation and incorporation of about 0.8 mol of reagent/mol of average enzyme subunit [Bednar, R.A., Hartman, F.C., & Colman, R.F. (1982) Biochemistry 21, 3681-3689]. Protection against inactivation is provided by isocitrate and Mn2+. We have now identified a critical modified peptide by comparison of the peptides labeled by BrKG at pH 6.1 in the absence and presence of isocitrate and Mn2+. Modified enzyme, isolated from unreacted BrKG, was incubated with [3H]NaBH4 to reduce the keto group of protein-bound 2-ketoglutarate and thereby introduce a radioactive tracer into the modified amino acid. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated by reverse-phase HPLC, first in 0.1% trifluoroacetic acid with a gradient in acetonitrile and then in 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas-phase sequencing gave the modified peptide: Ser-Ala-X-Val-Pro-Val-Asp-Phe-Glu-Glu-Val-Val-Val-Ser-Ser-Asn-Ala-Asp-Gl u-Glu- Asp-Ile-Arg. The corresponding tryptic peptide that was isolated from unmodified enzyme yielded the same sequence except for (carboxymethyl)cysteine at position 3, suggesting that cysteine is the target of 3-bromo-2-ketoglutarate. Pig heart NAD+-dependent isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma) that can be separated by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The peptide modified by 3-bromo-2-ketoglutarate, which is in or near the substrate site, is derived only from the separated gamma subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

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3-Bromo-2-ketoglutarate: a substrate and affinity label for DPN-dependent isocitrate dehydrogenase

Cited by 13 publications

References 35 publications

Affinity labeling of nicotinamide adenine dinucleotide-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate. Evidence for the formation of an unusual reaction product

Affinity labeling of nicotinamide adenine dinucleotide-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate. Evidence for the formation of an unusual reaction product

Molecular cloning and deduced amino acid sequences of the α- and β- subunits of mammalian NAD+-isocitrate dehydrogenase

Cysteinyl peptide labeled by 3-bromo-2-ketoglutarate in the active site of pig heart NAD+-dependent isocitrate dehydrogenase

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