β-Sulfur substituted α-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes

Abstract

The RS-isomers of β-mercapto-α-ketoglutarate, β-methylmercapto-α-ketoglutarate and β-methylmercapto-α-hydroxyglutarate have been synthesized. β-Mercapto-α-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP +, of the mitochondrial NADP-specific isozyme from pig heart ( K i = 5 nM; K m( DL-isocitrate) K i(RS-β- mercapto-α-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I 0·5 = 23 n m), and the NADP-linked enzymes from yeast ( K i = 58 nM) and Escherichia coli ( K i = 58 nM) at pH 7.4 and with Mg 2+ as activator. β-Mercapto-α-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. β-Mercapto-α-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase ( K i = 520 nM) than for the NADP-specific enzyme. β-Methylmercapto-α-ketoglutarate (I 0·5 = 10 μ m) was a much less effective inhibitor than the β-mercapto derivative for heart NADP-isocitrate dehydrogenase. The β-sulfur substituted α-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO 2. β-Methylmercapto-α-hydroxyglutarate, the expected product of reduction of β-methylmercapto-α-ketoglutarate, did not cause reduction of NADP + but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The β-sulfur substituted α-ketoglutarate derivatives were alternate substrates for α-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.