Removal of Metal from Carboxypeptidase A Proceeds via a Split Pathway: Implications for the General Mechanisms of Metalloenzyme Inactivation by Chelating Agents.
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引用次数: 0
Abstract
The chelation of protein-bound metal ions is typically thought to follow either a dissociative (D) or an associative (A) path. While the former mechanism involves the spontaneous dissociation of the metal from the protein prior to chelation, the latter route is characterized by the formation of an intermediate protein-metal-chelator ternary complex. Using the prototypical zinc protease carboxypeptidase A (CPA) and a variety of charged and neutral chelating agents, we demonstrate that inactivation of the enzyme (and likely other metalloproteins) proceeds through a split pathway with contributions from both D- and A-type mechanisms. In the case of charged chelators such as ethylenediaminetetraacetic acid (EDTA), the proportions of both paths could be tuned over a wide range through variation of the chelator concentration and the ionic strength, I (from ∼95% A type at low I values to ∼5% at high I values). By measuring the EDTA concentration and time dependence of CPA inactivation and fitting the obtained kinetic data to a modified time-dependent inhibition model, we obtained the rate constants for the A and D paths (kinact and koff, respectively) and the inhibition constant (KI) for the formation of the CPA-Zn2+-EDTA ternary complex, indicating that the decreased contribution of the A-type mechanism at high ionic strengths originates from a large (40-fold; at I = 0.5 M) increase in KI. This observation might be related to a triarginine motif in CPA that electrostatically steers negatively charged substrates into the active site and may therefore also guide carboxylate-bearing chelators toward the Zn2+ ion.
蛋白质结合的金属离子的螯合通常被认为遵循解离(D)或结合(A)途径。前一种机制涉及金属在螯合之前自发地从蛋白质中解离,而后一种途径的特点是形成中间的蛋白质-金属-螯合剂三元复合物。我们利用原型锌蛋白酶羧肽酶 A(CPA)和各种带电和中性螯合剂,证明了该酶(可能还有其他金属蛋白)的失活是通过一种分裂途径进行的,其中既有 D 型机制的贡献,也有 A 型机制的贡献。在乙二胺四乙酸(EDTA)等带电螯合剂的情况下,通过改变螯合剂浓度和离子强度 I(低 I 值时 A 型占 95%,高 I 值时占 5%),可以在很大范围内调整两种途径的比例。通过测量 CPA 失活的 EDTA 浓度和时间依赖性,并将获得的动力学数据拟合到改进的时间依赖性抑制模型,我们得到了 A 和 D 路径的速率常数(分别为 kinact 和 koff)以及 CPA-Zn2+-EDTA 三元复合物形成的抑制常数(KI)。这一观察结果可能与 CPA 中的一个三精氨酸基团有关,该基团能静电引导带负电荷的底物进入活性位点,因此也可能引导含羧酸的螯合剂向 Zn2+ 离子移动。
期刊介绍:
Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.