Purification and Characterization of NADP-Linked Isocitrate Dehydrogenase from the Copper-tolerant Wood-rotting Basidiomycete Fomitopsis palustris

Abstract

  NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), a key enzyme of the tricarboxylic acid cycle, was purified 672-fold as a nearly homogeneous protein from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris. The purified enzyme, with a molecular mass of 115 kDa, consisted of two 55-kDa subunits, and had the K_m of 12.7, 2.9, and 23.9 μM for isocitrate, NADP, and Mg²⁺, respectively, at the optimal pH of 9.0. The enzyme had maximum activity in the presence of Mg²⁺, which also helped to prevent enzyme inactivation during the purification procedures and storage. The enzyme activity was competitively inhibited by 2-oxoglutarate (K_i, 127.0 μM). Although adenine nucleotides and other compounds, including some of the metabolic intermediates of glyoxylate and tricarboxylic acid cycles, had no or only slight inhibition, a mixture of oxaloacetate and glyoxylate potently inhibited the enzyme activity and the inhibition pattern was a mixed type.