Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for alpha-ketoglutarate binding sites.

ABSTRACT

The interaction of oxidized nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent's potential as a selective reagent for alpha-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentrations of regent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and Kinact for bromoketoglutarate is 250 microM. Isocitrate and NADP+ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (Ki = 100 microM) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a Km of 250 microM and a Vmax comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (Ki = 3 microM) and ketoglutarate (Ki = 50 microM). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions, and shows absolute specificity for NADPH. However, since CO2 is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylation of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed. Commercial isocitrate dehydrogenase is purified 12-fold by affinity chromatography on thiol-agarose alkylated by bromoketoglutarate.