Homologous binding sites in yeast isocitrate dehydrogenase for cofactor (NAD+) and allosteric activator (AMP)

An Ping Lin, Lee McAlister-Henn

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an allosterically regulated octameric enzyme composed of two types of homologous subunits designated IDH1 and IDH2. Based on sequence comparisons and structural models, both subunits are predicted to have adenine nucleotide binding sites. This was tested by alanine replacement of residues in putative sites in each subunit. Targets included adjacent aspartate/isoleucine residues implicated as important for determining cofactor specificity in related dehydrogenases and a residue in each IDH subunit in a position occupied by histidine in other cofactor binding sites. The primary kinetic effects of D286A/I287A and of H281A replacements in IDH2 were found to be a dramatic reduction in apparent affinity of the holoenzyme for NAD+ and a concomitant reduction in Vmax. Ligand binding assays also showed that the H281A mutant enzyme fails to bind NAD+ under conditions that are saturating for the wild-type enzyme. In contrast, the primary effect of corresponding D279A/D280A and of R274A replacements in IDH1 is a reduction in holoenzyme binding of AMP, with concomitant alterations in kinetic and isocitrate binding properties normally associated with activation by this allosteric effector. These results suggest that the nucleotide cofactor binding site is primarily contributed by the IDH2 subunit, whereas the homologous nucleotide binding site in IDH1 has evolved for regulatory binding of AMP. These results are consistent with previous studies demonstrating that the catalytic isocitrate binding sites are comprised of residues primarily contributed by IDH2, whereas sites for regulatory binding of isocitrate are contributed by analogous residues of IDH1. In this study, we also demonstrate that a prerequisite for holoenzyme binding of NAD+ is binding of isocitrate/Mg2+ at the IDH2 catalytic site. This is comparable to the dependence of AMP binding upon binding of isocitrate at the IDH1 regulatory site.

Original languageEnglish (US)
Pages (from-to)12864-12872
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number15
DOIs
StatePublished - Apr 11 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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