N-ethylmaleimide profiling of yeast NADP-dependent isocitrate 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 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, NAPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-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 Cys269 is located in the region of the metal-isocitrate binding site.
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Y C
YC
Huang
R J
RJ
Haselbeck
L
L
McAlister-Henn
R F
RF
Colman
D000595:Amino Acid Sequence; D001665:Binding Sites; D005033:Ethylmaleimide / Q000494:pharmacology*; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D007523:Isocitrates / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015003:Yeasts / Q000201:enzymology*
D007523:Isocitrates; D010446:Peptide Fragments; D008345:Manganese; D009249:NADP; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D005033:Ethylmaleimide