Turnbull J, Cleland W W, Morrison J F
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Biochemistry. 1990 Nov 6;29(44):10245-54. doi: 10.1021/bi00496a014.
The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase-prephenate dehydrogenase, has been isolated from extracts of a plasmid-containing strain of Escherichia coli K12 and purified to homogeneity by a modified procedure that involves chromatography on both Matrex Blue A and Sepharose-AMP. Detailed studies of the dehydrogenase reaction have been undertaken with analogues of prephenate that act as substrates. The analogues, which included two of the four possible diastereoisomers of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate (deoxodihydroprephenate) as well as D- and L-arogenate, were synthesized chemically. As judged by their V/K values, all analogues were poorer substrates than prephenate. The order of their effectiveness as substrates is prephenate greater than one isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than L-arogenate greater than other isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than D-arogenate. Thus the dehydrogenase activity is dependent on the degree and position of unsaturation in the ring structure of prephenate as well as on the type of substitution on the pyruvyl side chain. With prephenate as a substrate, the reaction is irreversible because it involves oxidative decarboxylation. By contrast, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate undergoes only a simple oxidation, and thus, with this substrate, the reaction is reversible. Steady-state velocity data, obtained by varying substrates over a range of higher concentrations, suggest that the dehydrogenase reaction conforms to a rapid equilibrium, random mechanism with 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate as a substrate in the forward reaction or with the corresponding ketone derivative as a substrate in the reverse direction. The initial velocity patterns obtained by varying prephenate or 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate over a range of lower concentrations, at different fixed concentrations of NAD, were nonlinear and consistent with a unique model that is described by a velocity equation which is the ratio of quadratic polynomials. An equilibrium constant of 1.4 x 10(-7) M for the reaction in the presence of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate indicates that the equilibrium lies very much in favor of ketone production.
参与酪氨酸生物合成的双功能酶——分支酸变位酶-预苯酸脱氢酶，已从含质粒的大肠杆菌K12菌株提取物中分离出来，并通过一种改进方法纯化至同质，该方法包括在Matrex Blue A和琼脂糖-AMP上进行色谱分离。已使用作为底物的预苯酸类似物对脱氢酶反应进行了详细研究。这些类似物包括1-羧基-4-羟基-2-环己烯-1-丙酸（脱氧二氢预苯酸）四种可能的非对映异构体中的两种，以及D-和L-阿洛酸，是通过化学合成的。根据它们的V/K值判断，所有类似物作为底物都比预苯酸差。它们作为底物的有效性顺序为：预苯酸大于1-羧基-4-羟基-2-环己烯-1-丙酸的一种异构体大于L-阿洛酸大于1-羧基-4-羟基-2-环己烯-1-丙酸的其他异构体大于D-阿洛酸。因此，脱氢酶活性取决于预苯酸环结构中不饱和度的程度和位置以及丙酮酰侧链上取代基的类型。以预苯酸为底物时，该反应是不可逆的，因为它涉及氧化脱羧。相比之下，1-羧基-4-羟基-2-环己烯-1-丙酸仅发生简单氧化，因此，以该底物时，反应是可逆的。通过在一系列较高浓度下改变底物获得的稳态速度数据表明，以1-羧基-4-羟基-2-环己烯-1-丙酸为底物进行正向反应或以相应的酮衍生物为底物进行反向反应时，脱氢酶反应符合快速平衡随机机制。在不同固定浓度的NAD下，通过在一系列较低浓度下改变预苯酸或1-羧基-4-羟基-2-环己烯-1-丙酸获得的初始速度模式是非线性的，并且与一个独特的模型一致，该模型由一个速度方程描述，该方程是二次多项式的比值。在存在1-羧基-4-羟基-2-环己烯-1-丙酸的情况下，该反应的平衡常数为1.4×10⁻⁷ M，这表明平衡非常有利于酮的生成。
