Basis for half-site ligand binding in yeast NAD +-specific isocitrate dehydrogenase

An Ping Lin, Lee McAlister-Henn

Resultado de la investigación: Articlerevisión exhaustiva

2 Citas (Scopus)


Yeast NAD +-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD + binding sites, and four AMP binding sites, only half of the sites for each ligand can be measured in binding assays. On the basis of a potential interaction between side chains of Cys-150 residues in IDH2 subunits in each tetramer of the enzyme, ligand binding assays of wild-type (IDH1/IDH2) and IDH1/IDH2 C150S octameric enzymes were conducted in the presence of dithiothreitol. These assays demonstrated the presence of eight isocitrate and four AMP binding sites for the wild-type enzyme in the presence of dithiothreitol and for the IDH1/IDH2 C150S enzyme in the absence or presence of this reagent, suggesting that interactions between sulfhydryl side chains of IDH2 Cys-150 residues limit access to these sites. However, only two NAD + sites could be measured for either enzyme. A tetrameric form of IDH (an IDH1 G15D/IDH2 mutant enzyme) demonstrated half-site binding for isocitrate (two sites) in the absence of dithiothreitol and full-site binding (four sites) in the presence of dithiothreitol. Only one NAD + site could be measured for the tetramer under both conditions. In the context of the structure of the enzyme, these results suggest that an observed asymmetry between heterotetramers in the holoenzyme contributes to interactions between IDH2 Cys-150 residues and to half-site binding of isocitrate, but that a form of negative cooperativity may limit access to apparently equivalent NAD + binding sites.

Idioma originalEnglish (US)
Páginas (desde-hasta)8241-8250
Número de páginas10
EstadoPublished - sept 27 2011
Publicado de forma externa

ASJC Scopus subject areas

  • Biochemistry


Profundice en los temas de investigación de 'Basis for half-site ligand binding in yeast NAD +-specific isocitrate dehydrogenase'. En conjunto forman una huella única.

Citar esto