Park L S, Datta P
J Biol Chem. 1979 Aug 25;254(16):7927-34.
The activity of biodegradative threonine dehydratase of Escherichia coli K12 was reversibly inhibited by glyoxylate in the presence of AMP. Kinetic analysis showed that the inhibition was mixed with respect to L-threonine and competitive in terms of AMP; the inhibitory effect of glyoxylate was less pronounced at high protein concentrations. Incubation of dehydratase with L-threonine shifted the absorption maximum of the enzyme-bound pyridoxal phosphate from 413 to 425 nm; addition of glyoxylate completely prevented the threonine-mediated spectral shift. In addition to the inhibitory effect, incubation of purified enzyme with glyoxylate resulted in a progressive, irreversible inactivation of the enzyme and formation of inactive protein aggregates. The rates of inactivation were decreased with increasing concentrations of protein and AMP. During inactivation by glyoxylate, the 413-nm absorption maximum of the native enzyme was replaced by a new peak at 385 nm. Experiments with [14C]glyoxylate showed a rapid binding of 1 mol of glyoxylate per 147,000 g followed by a slow binding of 3 additional mol of glyoxylate; the glyoxylate-protein linkage was stable to acid precipitation and protein denaturants. Competition binding experiments revealed that pyruvate (which also inactivated the E. coli enzyme, Feldman, D.A., and Datta, P. (1975) Biochemistry 14, 1760-1767) did not interfere with the binding of glyoxylate or vice versa, suggesting that the two keto acids may occupy separate sites on the enzyme molecule. Nevertheless, experiments on enzyme inactivation using glyoxylate plus pyruvate reveal mutual interactions between these ligands in terms of lack of additive effect, retardation in the spectral shift due to glyoxylate, and stabilization of the enzyme in the presence and absence of AMP. We conclude from these results that the control of biodegradative threonine dehydratase is governed by a complex set of regulatory events resulting from reversible and irreversible association of these effectors with the enzyme molecule.
在AMP存在的情况下，乙醛酸可使大肠杆菌K12的生物降解型苏氨酸脱水酶的活性发生可逆抑制。动力学分析表明，这种抑制作用对L-苏氨酸而言属于混合型抑制，而对AMP来说则是竞争性抑制；在高蛋白浓度下，乙醛酸的抑制作用不太明显。将脱水酶与L-苏氨酸一起温育，会使酶结合的磷酸吡哆醛的最大吸收波长从413nm移至425nm；加入乙醛酸则完全阻止了苏氨酸介导的光谱位移。除了抑制作用外，将纯化的酶与乙醛酸一起温育会导致酶逐渐发生不可逆失活，并形成无活性的蛋白质聚集体。失活速率随着蛋白质和AMP浓度的增加而降低。在乙醛酸导致失活的过程中，天然酶在413nm处的最大吸收峰被385nm处的一个新峰所取代。用[14C]乙醛酸进行的实验表明，每147,000g酶快速结合1mol乙醛酸，随后又缓慢结合另外3mol乙醛酸；乙醛酸与蛋白质的连接对酸沉淀和蛋白质变性剂具有稳定性。竞争结合实验表明，丙酮酸（它也会使大肠杆菌的这种酶失活，费尔德曼，D.A.，和达塔，P.（1975年）《生物化学》14卷，1760 - 1767页）不会干扰乙醛酸的结合，反之亦然，这表明这两种酮酸可能占据酶分子上不同的位点。然而，使用乙醛酸加丙酮酸进行的酶失活实验表明，这些配体之间在缺乏加和效应、乙醛酸导致的光谱位移延迟以及在有无AMP存在时酶的稳定性方面存在相互作用。我们从这些结果得出结论，生物降解型苏氨酸脱水酶的调控是由这些效应物与酶分子的可逆和不可逆结合所导致的一系列复杂调控事件所控制的。
