Schneider G, Zeppezauer M
J Inorg Biochem. 1983 Feb;18(1):59-69. doi: 10.1016/0162-0134(83)85040-5.
The kinetics of the recombination of the metal-depleted active site of horse liver alcohol dehydrogenase (LADH) with metal ions have been studied over a range of pH and temperature. The formation rates were determined optically, by activity measurements, or by using the pH change during metal incorporation with a pH-indicator as monitor. The binding of Zn2+, Co2+, and Ni2+ ions occurs in a two-step process. The first step is a fast equilibrium reaction, characterized by an equilibrium constant K1. The spectroscopic and catalytic properties of the native or metal-substituted protein are recovered in a slow, monomolecular process with the rate constant k2. The rate constants k2 5.2 X 10(-2) sec-1 (Zn2+), 1.1 X 10(-3) sec-1 (Co2+), and 2 X 10(-4) sec-1 (Ni2+). The rate constants increase with increasing pH. Using temperature dependence, the activation parameters for the reaction with Co2+ and Ni2+ were determined. Activation energies of 51 +/- 2.5 kJ/mol (0.033 M N-Tris-(hydroxymethyl)methyl-2-aminomethane sulfonic acid (TES), pH 6, 9) for Co2+ and 48.5 +/- 4 kJ/mol (0.033 M TES, pH 7, 2) for Ni2+ at 23 degrees C were found. The correspondent activation entropies are - 146 +/- 10 kJ/mol K for Co2+ and - 163 +/- 9 kJ/mol K for Ni2+. Two protons are released during the binding of Zn2+ to H4Zn(n)2 LADH in the pH range 6.8-8.1. The binding of coenzyme, either reduced or oxidized, prevents completely the incorporation of metal ions, suggesting that the metal ions enter the catalytic site via the coenzyme binding domain and not through the hydrophobic substrate channel.
在一定的pH值和温度范围内，研究了马肝醇脱氢酶（LADH）金属耗尽活性位点与金属离子的重组动力学。通过光学方法、活性测量或使用pH指示剂监测金属掺入过程中的pH变化来确定形成速率。Zn2+、Co2+和Ni2+离子的结合分两步进行。第一步是快速平衡反应，其特征在于平衡常数K1。天然或金属取代蛋白的光谱和催化特性在一个缓慢的单分子过程中恢复，速率常数为k2。速率常数k2分别为5.2×10(-2)秒-1（Zn2+）、1.1×10(-3)秒-1（Co2+）和2×10(-4)秒-1（Ni2+）。速率常数随pH值升高而增加。利用温度依赖性，确定了与Co2+和Ni2+反应的活化参数。在23℃时，Co2+的活化能为51±2.5 kJ/mol（0.033 M N-三（羟甲基）甲基-2-氨基甲磺酸（TES），pH 6,9），Ni2+的活化能为48.5±4 kJ/mol（0.033 M TES，pH 7,2）。相应的活化熵分别为-146±10 kJ/mol K（Co2+）和-163±9 kJ/mol K（Ni2+）。在pH值6.8-8.1范围内，Zn2+与H4Zn(n)2 LADH结合时释放两个质子。辅酶无论是还原态还是氧化态，其结合都完全阻止了金属离子的掺入，这表明金属离子是通过辅酶结合域进入催化位点的，而不是通过疏水底物通道。
