Giannini I, Baroncelli V, Boccalon G, Fasella P
Eur J Biochem. 1976 Dec 11;71(2):475-81. doi: 10.1111/j.1432-1033.1976.tb11136.x.
The kinetics of the interaction between deionized supernatant aspartic aminotransferase and various anions (cacodylate, phosphate and chloride) were studied by the temperature-jump technique. The anion concentration in the range covered by our experiments does not affect the transamination rate. On the other hand the conformational transition, recently observed at the active site of the enzyme, is hindered by an excess of anions. A single relaxation effect was observed at the enzyme chromophore wavelength in systems containing the aldimine form of the enzyme and the above anions. It is shown that this effect corresponds to the protonation of the chromophore. The relaxation times were of about 10 mus with phosphate, 20-100 mus with cacodylate and 1-2 ms with chloride. The pH and concentration dependence of this effect were studied. The fits of experimental data to a rate equations for various models were tested by a chi2 analysis. The best fit was obtained with models where anions bind rapidly to a site close to the chromophore, so that the pK of the chromophore is affected by anions binding. The rate of the observed relaxation considerably increased when the anion has buffering capacities; this indicates, in the case of cacodylate and phosphate, that the acidic component of the buffer directly exchanges a proton with the enzyme chromophore.
采用温度跃升技术研究了去离子上清天冬氨酸转氨酶与各种阴离子（二甲胂酸盐、磷酸盐和氯化物）之间相互作用的动力学。我们实验所涵盖的阴离子浓度范围不影响转氨速率。另一方面，最近在酶活性位点观察到的构象转变受到过量阴离子的阻碍。在含有酶的醛亚胺形式和上述阴离子的体系中，在酶发色团波长处观察到单一的弛豫效应。结果表明，这种效应对应于发色团的质子化。二甲胂酸盐体系的弛豫时间约为10微秒，磷酸盐体系为20 - 100微秒，氯化物体系为1 - 2毫秒。研究了这种效应的pH值和浓度依赖性。通过卡方分析测试了实验数据对各种模型速率方程的拟合情况。在阴离子快速结合到靠近发色团的位点，从而使发色团的pK受阴离子结合影响的模型中获得了最佳拟合。当阴离子具有缓冲能力时，观察到的弛豫速率显著增加；在二甲胂酸盐和磷酸盐的情况下，这表明缓冲剂的酸性成分直接与酶发色团交换质子。
