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

Abstract

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 α2βγ. We have separated the subunits from unmodified and 8-BDBT[2-3H]NAD-modified enzymes by HPLC on a C4reverse-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 α and γ 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 α-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 α-subunit (amino acids 84–177) as well as one from the γ-subunit (amino acids 67–186). In the α-subunit, decreased modification by [7-14C]phenylglyoxal of Arg88and Arg98after 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 α subunit's Arg88and Arg98are both at or near the allosteric NADPH sites of the pig heart isocitrate dehydrogenase.