SUBUNIT LOCATION AND SEQUENCES OF THE CYSTEINYL PEPTIDES OF PIG-HEART NAD-DEPENDENT ISOCITRATE DEHYDROGENASE

Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of α2βγ, with the α subunit exhibiting a molecular weight of 39 000 and the β and γ each having molecular weights of 41 000. The amino-terminal sequences (33–35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the α subunit by 11–12 amino acids relative to that of the larger β and γ subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the α subunit. The β and y subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the β and γ subunits. The three subunits appear to be closely related, but β and γ are more similar to each other than either is to the α subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41–69], It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the αβ and αγ dimers are nonidentical but functionally similar. © 1990, American Chemical Society. All rights reserved.