Isocitrate Binding at Two Functionally Distinct Sites in Yeast NAD+-specific Isocitrate Dehydrogenase

An-Ping Lin,Lee Mcalister-Henn

doi:10.1074/jbc.m202534200

Abstract

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.

Highlights

Suggested significant cooperativity in subunit interactions and complex interdependent interactions of the enzyme with various ligands
Analysis of isocitrate binding by the wild-type enzyme was conducted in the absence or presence of 100 ␮M AMP, the allosteric activator of isocitrate dehydrogenase (IDH), to compare effects on S0.5 values with those obtained in kinetic analyses
Ligand-binding studies described in this report confirm previous predictions based on kinetic analyses of mutant enzymes that yeast IDH contains two types of isocitrate-binding sites (Fig. 2)

Summary

EXPERIMENTAL PROCEDURES

Host Yeast Strain and Plasmid Constructions—Wild-type and mutant forms of IDH were expressed in a yeast strain, IDH⌬12L (MAT␣ ade can100 his leu112 trp ura3–1 ⌬idh1::LEU2 ⌬idh2::HIS3), containing deletion/disruption mutations in chromosomal IDH1 and IDH2 loci [28]. Enzyme Expression and Purification—For purification of wild-type and mutant forms of IDH, transformant colonies were recovered from plates by growth for 16 h in 10 ml of YP medium (1% yeast extract, 2% Bacto-peptone) containing 2% glucose, transferred and grown for 24 h in 100 ml of YNB glucose medium lacking uracil to select for plasmid replication. The purified enzymes were stored at 4 °C in affinity column elution buffer (50 mM sodium phosphate, pH 7.5, 300 mM NaCl, and 200 mM imidazole) prior to kinetic or ligand-binding assays, which were performed within 24 h following purification. Binding is expressed as moles of bound ligand/mol of IDH holoenzyme (molecular weight ϭ 303,024), and each value represents an average from two independent experimental determinations

RESULTS

TABLE I Isocitrate binding parameters for yeast isocitrate dehydrogenases

Assays with the

DISCUSSION

Binding sites