Flavin conformational changes in the catalytic cycle of p-hydroxybenzoate hydroxylase substituted with 6-azido- and 6-aminoflavin adenine dinucleotide.
Flavin conformational changes in the catalytic cycle of p-hydroxybenzoate hydroxylase substituted with 6-azido- and 6-aminoflavin adenine dinucleotide.
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被 6-叠氮基和 6-氨基黄素腺嘌呤二核苷酸取代的对羟基苯甲酸羟化酶催化循环中的黄素构象变化。
DOI：
10.1021/bi971427u
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Massey,V
中科院分区：
文献类型：
--
作者：
Palfey,BA;Ballou,DP;Massey,V
晶体学研究已经证明了对羟基苯甲酸羟化酶（PHBH）的两种黄素构象[Gatti，D. L.，苍白，B。一、Lah，M.美国，Entsch，B.，Massey，V.，D.C.，D. P.的人，& Ludwig，M. L.（1994）《科学》266，110 - 114。Schreuder，H.一、Mattevi，A.，Obmolova，G.，Kalk，K. H、Hol，W. G. J.，货车德博尔特，F. J.T.，&货车Berkel，W. J. H.（1994）Biochemistry33，10161 - 10170]。一种构象的异咯嗪环系统（“外”构象）明显更多地暴露于溶剂，并且不处于必要的催化反应的位置，但是当天然底物与酶结合时，异咯嗪处于其化学功能的正确位置（“内”构象）。在这项研究中，构象变化在催化中的功能的几个方面进行了探索，使用野生型和Tyr 222 Phe形式的PHBH取代6-叠氮基FAD。这种黄素既是光谱探针又是光标记。含6-叠氮基FAD的酶是对羟基苯甲酸羟基化反应的一种较有效的催化剂。然而，中间体还原的6-叠氮基酶是化学不稳定的，和一小部分转化为6-氨基PHBH的消除N2在每个催化循环。NADPH对6-叠氮基FAD PHBH的还原几乎与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”到“外”构象的运动先于黄素还原。辐射的6-叠氮基PHBH与可见光共价标记的脯氨酸293，活性位点残基，条件下，其中的黄素采用的“在”构象，而没有蛋白质标记的条件下，其中的黄素是“出”。标记的蛋白质交换底物，并被还原的NADPH比光解前慢得多。因此，得出结论，异咯嗪运动需要吡啶核苷酸获得访问的活性位点和芳香族配体的交换。
Crystallographic studies have demonstrated two flavin conformations forp-hydroxybenzoate hydroxylase (PHBH) [Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., & Ludwig, M. L. (1994)Science266, 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)Biochemistry33, 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 ofp-hydroxybenzoate. However, the intermediate reduced 6-azido enzyme was chemically unstable, and a small fraction converted to 6-amino PHBH by the elimination of N2during 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.