N-Ethylmaleimide Profiling of Yeast NADP-Dependent Isocitrate Dehydrogenase

Yu Chu Huang, Robert J. Haselbeck, Lee McAlister-Henn, Roberta F. Colman

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30°C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NADPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-maleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: While no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

Original languageEnglish (US)
Pages (from-to)485-492
Number of pages8
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - Jan 1995


  • Chemical modification
  • Dehydrogenases
  • Isocitrate dehydrogenase
  • JV-ethylmaleimide

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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