Li Y, Harris W R
Department of Chemistry, University of Missouri-St. Louis, St. Louis, MO 63121, USA.
Biochim Biophys Acta. 1998 Sep 8;1387(1-2):89-102. doi: 10.1016/s0167-4838(98)00109-5.
The kinetics of iron removal from both forms of human serum monoferric transferrin by three ligands, 1, 2-dimethyl-3-hydroxypyridin-4-one (L1), 1-hydroxypyridin-2-one and acetohydroxamic acid, have been evaluated at pH 7.4 and 25.0 degreesC. In almost all cases the rate of iron removal follows simple saturation kinetics with respect to the ligand concentration. No spectroscopically distinct intermediates are observed during the iron removal reaction, which is consistent with a mechanism in which the rate-limiting step in iron removal is a protein conformational change. In the presence of chloride or perchlorate, most systems continue to follow simple saturation kinetics, but with significantly different kmax values. Chloride accelerates iron release from both transferrin binding sites, while perchlorate accelerates iron release from the C-terminal site but retards iron release from the N-terminal site. When the hydrochloride salt of L1 is used to prepare the L1 stock solution, the allosteric effect of the chloride produces a continuing increase in the rate of iron removal with increasing ligand concentration, so that one no longer observes simple saturation kinetics. A least squares fit of kobs vs. the ligand concentration for L1.HCl shows that the allosteric effect of the chloride not only enhances the first-order term for iron removal but also doubles the apparent kmax for the saturation term. This supports the view that allosteric binding of anionic ligands contributes to the observed variation in kmax among different ligands. A detailed description of this allosteric effect is not yet possible because the effect varies significantly from system to system, depending upon the specific anion that is binding at the allosteric site, the ligand that is used to remove the iron, and the transferrin lobe from which iron is removed.
在pH 7.4和25.0℃条件下，评估了三种配体1,2 - 二甲基 - 3 - 羟基吡啶 - 4 - 酮（L1）、1 - 羟基吡啶 - 2 - 酮和乙酰氧肟酸从两种形式的人血清单铁转铁蛋白中去除铁的动力学。在几乎所有情况下，铁去除速率相对于配体浓度遵循简单的饱和动力学。在铁去除反应过程中未观察到光谱上不同的中间体，这与铁去除的限速步骤是蛋白质构象变化的机制一致。在存在氯离子或高氯酸根离子的情况下，大多数体系继续遵循简单的饱和动力学，但具有显著不同的kmax值。氯离子加速了铁从两个转铁蛋白结合位点的释放，而高氯酸根离子加速了铁从C端位点的释放，但减缓了铁从N端位点的释放。当使用L1的盐酸盐制备L1储备溶液时，氯离子的变构效应导致随着配体浓度增加铁去除速率持续增加，因此不再观察到简单的饱和动力学。L1.HCl的观测速率常数kobs与配体浓度的最小二乘拟合表明，氯离子的变构效应不仅增强了铁去除的一级项，还使饱和项的表观kmax增加了一倍。这支持了阴离子配体的变构结合导致不同配体之间观察到的kmax变化的观点。由于这种变构效应因体系而异，取决于在变构位点结合的特定阴离子、用于去除铁的配体以及去除铁的转铁蛋白叶，因此目前还无法对这种变构效应进行详细描述。
