Two forms of ‘malic’ enzyme with different regulatory properties in Trypanosoma cruzi

Abstract

1. Cell-free extracts from culture epimastigotes of Trypanosoma cruzi contained two forms of NADP+-linked ‘malic’ enzyme (EC 1.1.1.40), I and II, with the same molecular weight but different electrophoretic mobilities and kinetic and regulatory properties. 2. The apparent Km for L-malate was lower for ‘malic’ enzyme I, with hyperbolic kinetics, whereas the kinetic pattern for ‘malic’ enzyme II was slightly sigmoidal (h 1.4). The kinetics for NADPH were hyperbolic for ‘malic’ enzyme I, and very complex for ‘malic’ enzyme II, suggesting both positive and negative co-operativity. 3. ‘Malic’ enzyme II was markedly inhibited by adenine nucleotides; AMP was the the most effective, at least in the presence of an excess of MnCl2. ‘Malic’ enzyme I was much less affected by the nucleotides. Both enzyme forms were inhibited by oxaloacetate, competitively towards L-malate, but the apparent Ki for ‘malic’ enzyme I (9 microM) was 10-fold lower than the value for ‘malic’ enzyme II. ‘Malic’ enzyme II, but not ‘malic’ enzyme I, was activated by L-aspartate and succinate (apparent Ka of 0.12 and 0.5 mM respectively); the activators caused a decrease in the apparent Km for L-malate and, to a lesser extent, in the apparent Km for NADP+. L-Aspartate, but not succinate, increased the apparent Vmax. 4. The inhibition by AMP suggests regulation by energy charge, with the L-malate-decarboxylation reaction catalysed by ‘malic’ enzyme II fulfilling a biosynthetic role. The inhibition by oxaloacetate and the activation by succinate are probably involved in the regulation of the ‘partial aerobic fermentation’ of glucose which yields succinate as final product. The activation by L-aspartate would facilitate the catabolism of this amino acid, when present in excess in the growth medium.