Valenzuela-Soto E M, Muñoz-Clares R A
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autonoma de Mexico, México City.
J Biol Chem. 1993 Nov 15;268(32):23818-23.
The kinetics of the oxidation of betaine aldehyde catalyzed by NAD(+)-betaine-aldehyde dehydrogenase, purified from amaranth leaves subjected to water deficit, were analyzed by steady state initial velocity and product and dead-end inhibition studies at low substrate concentrations. Only one product, NADH, gives inhibition. The other product of the reaction, glycine betaine, does not inhibit the enzyme even at concentrations as high as 10 mM. In dead-end inhibition experiments, AMP and choline were used as dead-end analogs of NAD+ and betaine aldehyde, respectively. The families of double-reciprocal plots in the range 0.010-0.500 mM NAD+ and 0.025-0.300 mM betaine aldehyde are linear and intersect at the left of the 1/v axis. NADH is a mixed inhibitor against NAD+ and betaine aldehyde. AMP is competitive with respect to NAD+ and mixed with betaine aldehyde. Choline is competitive against betaine aldehyde and uncompetitive with respect to NAD+. Our results are consistent with an Iso Ordered Bi Bi steady state mechanism in which NAD+ is the first substrate to bind to the enzyme and NADH is the last product to dissociate from it. To our knowledge, this is the first time that an Iso mechanism has been demonstrated by product inhibition studies, as predicted by Cleland (Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104-137).
从遭受水分亏缺的苋菜叶片中纯化得到的NAD(+)-甜菜碱醛脱氢酶催化甜菜碱醛氧化的动力学，通过低底物浓度下的稳态初速度、产物和终产物抑制研究进行分析。只有一种产物NADH产生抑制作用。该反应的另一种产物甘氨酸甜菜碱，即使在浓度高达10 mM时也不抑制该酶。在终产物抑制实验中，分别使用AMP和胆碱作为NAD+和甜菜碱醛的终产物类似物。在0.010 - 0.500 mM NAD+和0.025 - 0.300 mM甜菜碱醛范围内的双倒数图族是线性的，并且在1/v轴左侧相交。NADH是针对NAD+和甜菜碱醛的混合型抑制剂。AMP对NAD+具有竞争性，对甜菜碱醛为混合型。胆碱对甜菜碱醛具有竞争性，对NAD+为非竞争性。我们的结果与有序双双稳态机制一致，其中NAD+是第一个与酶结合的底物，NADH是最后一个从酶上解离的产物。据我们所知，这是首次通过产物抑制研究证明等机制，正如Cleland（Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104 - 137）所预测的那样。
