N-ETHYLMALEIMIDE PROFILING OF YEAST NADP-DEPENDENT ISOCITRATE DEHYDROGENASE

Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethy l-maleimide (NEM) at pH 7.7 and 30 degrees C. Reaction with cysteine(3 82) occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during whic h lysine(343) is the major target of NEM, while minor products result from reaction at cysteine(73) and cysteine(354). Protection against th e second phase of inactivation is provided by NADP, NADPH, or manganou s-isocitrate. Comparison of the time-dependence of inactivation and th e products of reaction with N-ethyl-maleimide (NEM profiling) of the p ig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 1491 8-14925) and yeast NADP-specific isocitrate dehydrogenases have been c oupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been re ached: while no cysteine is essential for activity, yeast Cys(382)/pig Cys(379) is close to the adenine portion of the NADP binding site, an d pig Cys(269) is located in the region of the metal-isocitrate bindin g site. (C) 1995 Academic Press, Inc.