N-ethylmaleimide profiling of yeast NADP-dependent isocritate dehydrogenase

Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30 degrees 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 maj...

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Bibliographic Details
Published inArchives of biochemistry and biophysics Vol. 316; no. 1
Main Authors Huang, Y.C. (University of Delaware, Newark, DE.), Haselbeck, R.J, McAlister-Henn, L, Colman, R.F
Format Journal Article
LanguageEnglish
Published 10.01.1995
Subjects
CISTEINA
COENZIMAS
COENZYME
CYSTEINE
INHIBIDORES DE ENZIMAS
INHIBITEUR D'ENZYME
ISOCITRATE DESHYDROGENASE
ISOCITRATO DESHIDROGENASA
LEVADURA
LEVURE
LISINA
LYSINE
NUCLEOTIDE
NUCLEOTIDOS
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Summary:Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30 degrees 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-ethylmaleimide (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 Cys239 is located in the region of the metal-isocitrate binding site
Bibliography:F60
9545779
ISSN:0003-9861
1096-0384