Vas M, Berni R, Batke J, Keleti T, Rossi G L
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest.
Arch Biochem Biophys. 1988 May 15;263(1):121-9. doi: 10.1016/0003-9861(88)90620-0.
Ammonium sulfate, a typical component of crystallization media of proteins, stabilizes an inactive conformation of pig muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, in the presence of ammonium sulfate the reconstitution of the catalytically active holoenzyme from the apoenzyme and NAD is not instantaneous, as in the case of enzymes from Bacillus stearothermophilus and the Mediterranean lobster Palinurus vulgaris. With pig muscle enzyme, at pH 6.0, the time course of formation of the characteristic Racker band can be monitored by a rapid mixing stopped flow technique. Activation follows a single exponential curve with a rate constant independent of the concentration of both NAD and protein and, therefore, appears to be limited by a slow protein isomerization (k = 7 +/- 2 s-1). Accordingly, when the apoenzyme is simultaneously exposed to NAD and either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate, the ensuing reactions (the redox and the acylation steps, respectively) are kinetically limited by the same protein isomerization. At pH 7.0 and 8.0, however, two among the four active sites react with NAD at an unmeasurably high rate, while the other two sites behave as they do at pH 6.0. When the pig muscle apoenzyme is preincubated and allowed to react with either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate before the rapid mixing with NAD, both the redox reaction and the NAD-dependent activation of apo-acyl-enzyme toward arsenolysis become unmeasurably fast. Similarly, when the sulfate in the medium is replaced by ions such as phosphate and citrate, the reconstitution of the active holoenzyme is practically instantaneous. Thus, the slow protein isomerization observed in the presence of sulfate and abolished by competing substrates and anions is diagnostic of a structural state of the pig muscle apoenzyme, which is induced by sulfate ions bound within the enzyme active site.
硫酸铵是蛋白质结晶介质的典型成分，它能稳定猪肌肉甘油醛-3-磷酸脱氢酶的无活性构象。事实上，在硫酸铵存在的情况下，从脱辅酶和NAD重构具有催化活性的全酶并非像嗜热脂肪芽孢杆菌和地中海龙虾黄斑龙虾的酶那样是即时的。对于猪肌肉酶，在pH 6.0时，特征性的拉克尔带形成的时间进程可以通过快速混合停流技术进行监测。激活遵循单一指数曲线，速率常数与NAD和蛋白质的浓度均无关，因此，似乎受缓慢的蛋白质异构化限制（k = 7 +/- 2 s-1）。相应地，当脱辅酶同时暴露于NAD和甘油醛-3-磷酸或1,3-二磷酸甘油酸时，随后的反应（分别为氧化还原步骤和酰化步骤）在动力学上受相同的蛋白质异构化限制。然而，在pH 7.0和8.0时，四个活性位点中的两个与NAD以极高的速率反应，而另外两个位点的行为与在pH 6.0时相同。当猪肌肉脱辅酶在与NAD快速混合之前预先孵育并使其与甘油醛-3-磷酸或1,3-二磷酸甘油酸反应时，氧化还原反应以及脱辅酶酰基酶对砷解作用的NAD依赖性激活都变得极快，以至于无法测量。同样，当培养基中的硫酸根被磷酸根和柠檬酸根等离子取代时，活性全酶的重构实际上是即时的。因此，在硫酸根存在下观察到的缓慢蛋白质异构化，并被竞争性底物和阴离子消除，这是猪肌肉脱辅酶一种结构状态的特征，这种结构状态是由结合在酶活性位点内的硫酸根离子诱导的。
