Cárdenas M L
Bioénergétique et Ingénierie des Protéines, Institut Fédératif "Biologie Structurale et Microbiologie," Centre National de la Recherche Scientifique, 31 Chemin Joseph-Aiguier, Marseille Cedex 20, 13402, France.
Methods. 2001 Jun;24(2):175-80. doi: 10.1006/meth.2001.1178.
Enzymes often act on more than one substrate, and the question then arises as to whether this can be attributed to the existence of two different enzymes that have not been separated or, more interesting, to the presence of two different active sites in the same enzyme. The competition plot is a kinetic method that allows us to test with little experimentation whether the two reactions occur at the same site or at different sites. It consists of making mixtures of the two substrates and plotting the total rate against a parameter p that defines the concentrations of the two substrates in terms of reference concentrations chosen to give the same rates at p = 0 and p = 1, i.e., when only one of the substrates is present. With a slight modification of the equations it can also be applied to enzymes that deviate from Michaelis-Menten kinetics. If the two substrates react at the same site, the competition plot gives a horizontal straight line; i.e., the total rate is independent of p. In contrast, if the two reactions occur at two separate and independent sites a curve with a maximum is obtained; separate reactions with cross-inhibition generate curves with either maxima or minima according to whether the Michaelis constants of the two substrates are smaller or larger than their inhibition constants in the other reactions. Strategies to avoid ambiguous results and to improve the sensitivity of the plot are described. A practical example is given to facilitate the experimental protocol for this plot.
酶常常作用于不止一种底物，于是问题就出现了：这是归因于两种尚未分离的不同酶的存在，还是更有意思地归因于同一种酶中存在两个不同的活性位点。竞争图是一种动力学方法，它让我们只需进行少量实验就能检验这两个反应是在同一位置还是在不同位置发生。它包括制备两种底物的混合物，并将总速率绘制成相对于参数p的图，p根据选定的参考浓度来定义两种底物的浓度，使得在p = 0和p = 1时（即仅存在一种底物时）速率相同。对这些方程稍作修改后，它也可应用于偏离米氏动力学的酶。如果两种底物在同一位置反应，竞争图会给出一条水平直线；即总速率与p无关。相反，如果两个反应在两个分开且独立的位置发生，则会得到一条有最大值的曲线；伴有交叉抑制的单独反应会根据两种底物的米氏常数在其他反应中小于还是大于它们的抑制常数而产生有最大值或最小值的曲线。文中描述了避免产生模糊结果以及提高该图灵敏度的策略。给出了一个实际例子以方便该图的实验方案。
