Saxena A, Hensley P, Osborne J C, Fleming P J
J Biol Chem. 1985 Mar 25;260(6):3386-92.
The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.
先前已证明，来自牛肾上腺髓质的可溶性多巴胺β-羟化酶以Mr = 290,000的四聚体形式存在，该四聚体由两个通过二硫键连接的二聚体组成。在此我们报告，这种酶还能发生可逆的四聚体-二聚体解离，且这种解离依赖于pH。在pH 5.0条件下对多巴胺β-羟化酶进行凝胶渗透色谱分析，其斯托克斯半径为5.8 nm。当pH值变为5.7时，斯托克斯半径变为6.9 nm。对纯化酶进行沉降平衡分析表明，分子大小的这种变化是由于分子量的改变。在低蛋白浓度下，该酶在pH 5.0时的估计Mr为145,000，而在高蛋白浓度下，在pH 5.7时接近290,000。Mr的这种变化与四聚体-二聚体解离的存在以及当pH从5.0增加到5.7时平衡常数从1.8×10⁻⁶ M变为1.16×10⁻⁹ M相一致。这种依赖于pH的亚基解离与酶活性的pH依赖性变化相关。纯化的牛可溶性多巴胺β-羟化酶活性在pH 5.0时是酪胺浓度的双曲线函数。然而，该羟化酶活性在pH 6.0时表现出非双曲线动力学。在pH 6.0时获得的动力学数据可以通过拟合一个模型来解释，该模型包含由四聚体酶向二聚体亚基的pH依赖性部分解离产生的两种不同催化形式的酶。这两种催化形式显然具有相同的最大速度；然而，它们对底物的米氏常数不同；二聚体形式的Km较低而四聚体形式的Km较高。由于牛肾上腺髓质嗜铬颗粒内部的pH约为5.5，我们得出结论，多巴胺β-羟化酶的亚基在生理上重要的pH范围内处于动态解离状态。
