McCoubrey W K, Maines M D
University of Rochester School of Medicine, Department of Biophysics, New York 14642.
Eur J Biochem. 1994 Jun 1;222(2):597-603. doi: 10.1111/j.1432-1033.1994.tb18902.x.
Biliverdin reductase is unique among all enzymes described to date in having two pH optima, 6.75 and 8.7, at which NADH or NADPH, respectively, are required for activity. The enzyme converts biliverdin to bilirubin in mammals. The mature enzyme, which is 293 amino acids long, has 3 cysteine residues, and is sulfhydryl dependent. To understand the role of the cysteine residues in enzyme activity, we examined the effects of the neutral substitution with alanine of each of three residues, individually and in combination, by site-directed mutagenesis. These residues in the predicted amino acid sequence of rat biliverdin reductase correspond to amino acids 73, 280 and 291. The modification of the amino-proximal cysteine (Cys73), which is flanked by a tyrosine residue, completely inactivated the enzyme with NADH at pH 6.75 and NADPH at pH 8.7. The loss of reductase activity was not due to changes in three-dimensional characteristics of the protein as suggested by its mobility in a non-denaturing gel. Although modification of either of the two cysteines located near the C-terminus (Cys280 and Cys291) significantly reduced activity with both cofactors, these mutations did not inactivate the enzyme. Comparison of Km values for the Cys280-->Ala and Cys291-->Ala mutants with the wild type protein, at pH 8.7, suggests that Cys280 principally functions in substrate binding while Cys291 is predominantly involved in cofactor binding. This assignment probably also applies at pH 6.75. Comparison of kcat of the mutants with wild type shows that mutation of Cys280 decreases Vmax of the enzyme. Mutation of both C-terminal cysteines caused inactivation of the enzyme, comparable to that produced by mutation of Cys73. Analysis by circular dichroism at far-ultraviolet wavelengths suggests that the alterations in activity are not the result of changes in the secondary structure of these mutants. These results are consistent with Cys73 having a central role in substrate/cofactor binding while biliverdin reductase can function, albeit at a reduced rate, with only one of the near C-terminus cysteines. The results are further consistent with the suggestion that although the two C-terminal cysteines have preferential affinities, they can serve similar functions in the interaction with substrate/cofactor.
迄今为止所描述的所有酶中，胆绿素还原酶独具特色，它有两个最适pH值，分别为6.75和8.7，在这两个pH值下，分别需要NADH或NADPH来发挥活性。该酶在哺乳动物体内将胆绿素转化为胆红素。成熟的酶由293个氨基酸组成，有3个半胱氨酸残基，且依赖巯基。为了了解半胱氨酸残基在酶活性中的作用，我们通过定点诱变分别及联合研究了将三个残基中的每一个用丙氨酸进行中性取代的效果。大鼠胆绿素还原酶预测氨基酸序列中的这些残基分别对应于第73、280和291位氨基酸。靠近氨基端的半胱氨酸（Cys73）两侧是一个酪氨酸残基，对其进行修饰后，该酶在pH 6.75时利用NADH以及在pH 8.7时利用NADPH的活性完全丧失。还原酶活性的丧失并非如非变性凝胶中其迁移率所表明的那样是由于蛋白质三维结构的改变。虽然靠近羧基端的两个半胱氨酸（Cys280和Cys291）中的任何一个发生修饰都会显著降低两种辅因子存在时的活性，但这些突变并未使酶失活。在pH 8.7时，将Cys280→Ala和Cys291→Ala突变体与野生型蛋白的Km值进行比较，结果表明Cys280主要在底物结合中起作用，而Cys291主要参与辅因子结合。这种分配在pH 6.75时可能也适用。将突变体与野生型的kcat进行比较表明，Cys280突变会降低酶的Vmax。两个羧基端半胱氨酸都发生突变会导致酶失活，其失活程度与Cys73突变所产生的失活程度相当。在远紫外波长下通过圆二色性分析表明，活性的改变并非这些突变体二级结构变化的结果。这些结果与Cys73在底物/辅因子结合中起核心作用一致，而胆绿素还原酶仅利用靠近羧基端的一个半胱氨酸时也能发挥作用，尽管速率会降低。这些结果还进一步支持了以下观点：虽然两个羧基端半胱氨酸具有优先亲和力，但它们在与底物/辅因子的相互作用中可发挥相似的功能。
