Voegele R T, Mitsch M J, Finan T M
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ont. L8S 4K1, Canada.
Biochim Biophys Acta. 1999 Jul 13;1432(2):275-85. doi: 10.1016/s0167-4838(99)00112-0.
The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.
革兰氏阴性菌苜蓿根瘤菌含有两种不同的苹果酸酶。我们报道了这两种同工酶的纯化至均一状态及其体外特性。两种酶均表现出异常高的亚基分子量，约为82 kDa。NAD(P)(+)特异性苹果酸酶[EC 1.1.1.39]对苹果酸表现出正协同性，但对辅因子NAD(+)或NADP(+)表现出米氏动力学行为。该酶受到底物抑制，并显示出乙酰辅酶A的变构调节作用，这种作用迄今为止仅在一些依赖NADP(+)的苹果酸酶中被描述过。其活性受到琥珀酸和富马酸的正调节。与NAD(P)(+)特异性苹果酸酶不同，依赖NADP(+)的苹果酸酶[EC 1.1.1.40]对苹果酸和NADP(+)表现出米氏动力学行为。除了产物抑制外，该酶不受到任何调节机制的影响。两种苹果酸酶均未检测到丙酮酸的还原羧化作用或草酰乙酸的脱羧作用。因此，我们对苜蓿根瘤菌两种苹果酸酶的特性描述表明了一些迄今为止所描述的苹果酸酶所不具备的特征。
