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°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.