Affinity purification and kinetic analysis of mutant forms of yeast NAD+-specific isocitrate dehydrogenase

Wen Ning Zhao, Lee McAlister-Henn

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Polyhistidine tags were added to the carboxyl termini of the two homologous subunits of yeast NAD+-specific isocitrate dehydrogenase (IDH). The tag in either the IDH1 or IDH2 subunit permits one-step affinity purification from yeast cellular extracts of catalytically active and allosterically responsive holoenzyme. This expression system was used to investigate subunit-specific contributions of residues with putative functions in adenine nucleotide binding. The primary effect of simultaneous replacement of the adjacent Asp-279 and Ile-280 residues in IDH1 with alanines is a dramatic loss of activation by AMP. In contrast, alanine replacement of the homologous Asp-286 and Ile-287 residues in IDH2 does not alter the allosteric response to AMP, but produces a 160-fold reduction in V(max) due to a 70-fold increase in the S0.5 value for NAD+. These results suggest that the targeted aspartate/isoleucine residues may contribute to regulator binding in IDH1 and to cofactor binding in IDH2, i.e. that these homologous residues are located in regions that have evolved for binding the adenine nucleotide components of different ligands. In other mutant enzymes, an alanine replacement of Asp-191 in IDH1 eliminates measurable catalytic activity, and a similar substitution of the homologous Asp-197 in IDH2 produces pleiotropic catalytic effects. A model is presented for the primary function of IDH2 in catalysis and of IDH1 in regulation, with crucial roles for these single aspartate residues in the communication and functional interdependence of the two subunits.

Original languageEnglish (US)
Pages (from-to)21811-21817
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number35
DOIs
StatePublished - Aug 29 1997

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Isocitrate Dehydrogenase
Alanine
NAD
Yeast
Purification
Adenine Nucleotides
Yeasts
Adenosine Monophosphate
Aspartic Acid
Kinetics
Holoenzymes
Isoleucine
Catalysis
Catalyst activity
Substitution reactions
Chemical activation
Ligands
Communication
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Affinity purification and kinetic analysis of mutant forms of yeast NAD+-specific isocitrate dehydrogenase. / Zhao, Wen Ning; McAlister-Henn, Lee.

In: Journal of Biological Chemistry, Vol. 272, No. 35, 29.08.1997, p. 21811-21817.

Research output: Contribution to journalArticle

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