Palfey B A, Ballou D P, Massey V
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Biochemistry. 1997 Dec 16;36(50):15713-23. doi: 10.1021/bi971427u.
Crystallographic studies have demonstrated two flavin conformations for p-hydroxybenzoate hydroxylase (PHBH) [Gatti, D. L., Palfey, B. A. , Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., & Ludwig, M. L. (1994) Science 266, 110-114. Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., & van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. The isoalloxazine ring system of one conformation (the "out" conformation) is significantly more exposed to solvent and is not in position for necessary catalytic reactions, but when the natural substrate is bound to the enzyme, the isoalloxazine is in the correct position (the "in" conformation) for its chemical function. In this study, several aspects of the function of the conformational change in catalysis were explored using the wild-type and Tyr222Phe forms of PHBH substituted with 6-azido FAD. This flavin served as both a spectral probe and a photolabel. The enzyme containing 6-azido FAD was a relatively effective catalyst for the hydroxylation of p-hydroxybenzoate. However, the intermediate reduced 6-azido enzyme was chemically unstable, and a small fraction converted to 6-amino PHBH by the elimination of N2 during each catalytic cycle. The reduction of 6-azido FAD PHBH by NADPH was almost as fast as the reduction of the natural enzyme. The characteristic spectral change caused by NADPH binding prior to hydride transfer strongly suggests that flavin movement from the "in" to the "out" conformation precedes flavin reduction. Irradiation of 6-azido PHBH with visible light covalently labeled proline 293, an active site residue, under conditions in which the flavin adopted the "in" conformation, while no protein labeling occurred under conditions in which the flavin was "out". The labeled protein exchanged substrate and was reduced by NADPH much more slowly than before photolysis. It is therefore concluded that isoalloxazine movement is required for pyridine nucleotide to gain access to the active site and for the exchange of aromatic ligands.
晶体学研究表明，对羟基苯甲酸羟化酶（PHBH）存在两种黄素构象[加蒂，D. L.，帕尔菲，B. A.，拉赫，M. S.，恩奇，B.，梅西，V.，巴卢，D. P.，& 路德维希，M. L.（1994年）《科学》266卷，第110 - 114页。施勒德，H. A.，马特维，A.，奥布莫洛娃，G.，卡尔克，K. H.，霍尔，W. G. J.，范德博尔特，F. J. T.，& 范伯克尔，W. J. H.（1994年）《生物化学》33卷，第10161 - 10170页]。一种构象（“外向”构象）的异咯嗪环系统显著更暴露于溶剂中，且处于无法进行必要催化反应的位置，但当天然底物与酶结合时，异咯嗪处于其化学功能所需的正确位置（“内向”构象）。在本研究中，使用野生型和用6 - 叠氮基FAD取代的Tyr222Phe形式的PHBH，探索了催化过程中构象变化功能的几个方面。这种黄素既作为光谱探针又作为光标记物。含有6 - 叠氮基FAD的酶是对羟基苯甲酸羟化反应相对有效的催化剂。然而，中间还原态的6 - 叠氮基酶在化学上不稳定，并且在每个催化循环中，一小部分通过消除N₂转化为6 - 氨基PHBH。NADPH对6 - 叠氮基FAD PHBH的还原几乎与对天然酶的还原一样快。在氢化物转移之前由NADPH结合引起的特征光谱变化强烈表明，黄素从“内向”构象向“外向”构象的移动先于黄素还原。在黄素采取“内向”构象的条件下，用可见光照射6 - 叠氮基PHBH可共价标记脯氨酸293（一个活性位点残基），而在黄素处于“外向”构象的条件下则没有蛋白质标记。标记后的蛋白质交换底物，并且被NADPH还原的速度比光解前慢得多。因此可以得出结论，异咯嗪的移动是吡啶核苷酸进入活性位点以及芳香配体交换所必需的。
