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.
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.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D007521:Isocitrate Dehydrogenase / Q000032:analysis* / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine
D005971:Glutamates; D010446:Peptide Fragments; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; C047570:2-((4-bromo-2,3-dioxobutyl)thio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)