Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants.
Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants.
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DOI：
10.1016/s0021-9258(19)47379-1
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发表时间：
1991-09
期刊：
影响因子：
--
通讯作者：
B. Entsch;B. Palfey;D. Ballou;Vincent Massey
中科院分区：
文献类型：
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作者：
B. Entsch;B. Palfey;D. Ballou;Vincent Massey
研究了黄素蛋白对羟基苯甲酸羟化酶催化反应中蛋白残基对底物活化的作用。x射线晶体学（Schreuder， H. A., Prick， P.A.J, Wieringa, r.k., Vriend， G., Wilson， K.S, Hol, w.g.j, and Drenth， J. (1989) J. Mol. Biol. 208, 679-696）表明tyr1 -201和tyr1 -385与对羟基苯甲酸酯的4-OH形成氢键网络。因此，构建了位点定向突变体，将这些酪氨酸转化为苯丙氨酸。光谱（可见光和荧光）特性、还原电位和结合常数与野生型非常相似，表明突变体没有发生重大的结构变化。在没有底物的情况下，突变体和野生型在FAD谱上表现出类似的ph依赖性变化。然而，Tyr-201 -Phe的酶-底物复合物缺乏在野生型和Tyr-385 -Phe中观察到的电离，其优先结合底物的酚酸形式。Tyr-201—Phe没有表现出偏好，表明Tyr-201是电离底物所必需的。突变体酶的活性低于野生型酶的6%。采用停流技术研究了对催化性能的影响。NADPH对FAD的还原速度较慢，在tyr1 -201 -Phe中减慢10倍，在tyr1 -385 -Phe中减慢100倍。当还原的tyr1 -201—ph -对羟基苯甲酸配合物与氧反应时，观察到一个长寿命的黄素- c (4a)-氢过氧化物，它缓慢地消除H2O2，几乎没有羟基化。因此，Tyr-201的作用是通过稳定酚酸来激活底物。Tyr-385—Phe与氧反应生成25%的氧化酶和75%的黄素氢过氧化物，成功地将底物羟基化。这个突变体也羟基化产物（3,4 -二羟基苯甲酸酯）形成没食子酸。
The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201—-Phe lacks an ionization observed in both wild type and Tyr-385—-Phe, which preferentially bind the phenolate form of substrates. Tyr-201—-Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201—-Phe and 100-fold in Tyr-385—-Phe. When the reduced Tyr-201—-Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385—-Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.