Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase.
Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octamer containing two types of homologous subunits. Ligand-binding analyses were conducted to examine effects of residue changes in putative catalytic and regulatory isocitrate-binding sites respectively contained in IDH2 and IDH1 subunits. Replacement of homologous serine residues in either subunit site, S98A in IDH2 or S92A in IDH1, was found to reduce by half the total number of holoenzyme isocitrate-binding sites, confirming a correlation between detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP. Replacement of both serine residues eliminates isocitrate binding and measurable catalytic activity. The putative isocitrate-binding sites of IDH1 and IDH2 contain five identical and four nonidentical residues. Reciprocal replacement of the four nonidentical residues in either or both subunits (A108R, F136Y, T241D, and N245D in IDH1 and/or R114A, Y142F, D248T, and D252N in IDH2) was found to be permissive for isocitrate binding. This provides further evidence for two types of binding sites in IDH, although the authentic residues have been shown to be necessary for normal kinetic contributions. Finally, the mutant enzymes with residue replacements in the IDH1 site were found to be unable to bind AMP, suggesting that allosteric activation is dependent both upon binding of isocitrate at the IDH1 site and upon the changes in the enzyme normally elicited by this binding.
Lin
An-Ping
AP
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
McAlister-Henn
Lee
L
D000249:Adenosine Monophosphate / Q000378:metabolism; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007523:Isocitrates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D007523:Isocitrates; D008024:Ligands; D009243:NAD; D000249:Adenosine Monophosphate; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase