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

Abstract

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)

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

“…Perhaps a distant relationship between these two isocitrate dehydrogenases is indicated. However, there is no resemblance between the cysteine (y subunit, peak VII) labeled by the substrate analogue 3-bromo-2-ketoglutarate in the active site of the pig heart NAD enzyme (Saha et al, 1989) and any of the cysteines of the E. coli enzyme, despite the fact that the bacterial enzyme is also inactivated by 3-bromo-2-ketoglutarate (Ehrlich and Colman, unpublished data). Other papers have reported inactivation of the NAD-dependent isocitrate dehydrogenase from pig heart by reaction of its cysteines with iodoacetate (Mauck & Colman, 1976) and from bovine heart by reaction of cysteines with 5,5'-dithiobis(2nitrobenzoate), TV-ethylmaleimide, and />mercuribenzenesulfonate (Fan & Plaut, 1974).…”

“…However, the complete amino acid sequence of the NADdependent isocitrate dehydrogenase is not yet known, and in only two cases have the chemically modified peptides been isolated and their sequences determined Saha et al, 1989). Further characterization of the subunits is essential in order to understand the relationship between the enzyme structure and its catalytic and regulatory sites.…”

Molecular cloning and deduced amino acid sequences of the γ-subunits of rat and monkey NAD+-isocitrate dehydrogenases

“…contribution of each subunit to the catalytic and regulatory properties of the mammalian holoenzyme is unknown. All three subunits are labelled by incubation with the isocitrate analogue 3-ene-2-oxoglutarate (Bednar and Colman, 1982), but a single peptide exclusive to the y-subunit was covalently modified by an alternative analogue, 3-bromo-2-oxoglutarate (Saha et al, 1989). Binding studies suggest that there is only one Ca2+-binding site per a2/Jy unit (Rutter and Denton, 1989b).…”

Natural and synthetic 2-oxoglutarate derivatives are substrates for oncogenic variants of human isocitrate dehydrogenase 1 and 2

“…For example, recent evidence suggests that extracellular 2HG is taken up by CD8-positive T cells in which it inhibits lactate dehydrogenase, thus altering glucose metabolism resulting in decreased proliferation, decreased cytokine production, and a decreased ability to kill target cells ( 25 ). Some 2-oxoacid and 2OG derivatives have been shown to be poor substrates of pig and rat WT IDH (obtained from homogenized pig heart and rat liver, respectively) ( 13 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ); however, the substrate selectivity of human oncogenic IDH1/2 variants with 2-oxoacids other than 2OG has not yet been investigated. Such studies are of interest because some 2OG derivatives are present in biology, including, for example, C3- and C4-alkylated 2OG derivatives in human food ( Fig.…”