Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline.
Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline.
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PutA 蛋白的构象变化和膜缔合与脯氨酸对其 FAD 辅因子的还原一致。
DOI：
10.1016/s0021-9258(18)52967-7
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
J. M. Wood
中科院分区：
文献类型：
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作者：
E. Brown;J. M. Wood
PutA蛋白既是put阻遏物又是具有脯氨酸和δ 1-吡咯啉-5-羧酸脱氢酶活性的膜结合酶。纯化的PutA蛋白与膜囊泡结合所需的条件表明，氧化还原转换机制可能决定PutA蛋白作为脱氢酶发挥功能的比例（Wood，J.M.等人（1987）Proc. Acad. sci. USA 84，373 - 377）。在中性pH下，用1 M KBr从PutA蛋白释放FAD辅因子。脱辅基蛋白保留δ 1-吡咯啉-5-羧酸脱氢酶和DNA结合，但不保留脯氨酸脱氢酶活性。用FAD重建完全恢复脯氨酸脱氢酶活性。脯氨酸在0.11 mM的浓度引起的一半最大漂白的FAD在PutA。胰凝乳蛋白酶消化的PutA蛋白质的存在和不存在的脯氨酸表明，持久性的119 kDa的蛋白质片段的特征是减少的蛋白质。相同的消化模式，从脱辅基蛋白的存在和不存在的脯氨酸。产生的119-kDa片段的量随脯氨酸浓度而变化，产生0.056 mM脯氨酸的中点。与膜囊泡相关的PutA蛋白的分数也是脯氨酸浓度的函数，产生0.10 mM脯氨酸的滴定中点。因此，膜结合与黄素减少和蛋白质构象的变化一致。
The PutA protein is both the put repressor and a membrane-bound enzyme with proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities. The conditions required for association of purified PutA protein with membrane vesicles suggested that a redox switching mechanism might determine the proportion of PutA protein functioning as a dehydrogenase (Wood, J. M. (1987) Proc. Natl. Acad. Sci. USA 84, 373-377). The FAD cofactor was released from the PutA protein with 1 M KBr at neutral pH. The apoprotein retained delta 1-pyrroline-5-carboxylate dehydrogenase and DNA binding but not proline dehydrogenase activity. Reconstitution with FAD fully restored proline dehydrogenase activity. Proline at a concentration of 0.11 mM caused half-maximal bleaching of the FAD in PutA. Chymotryptic digestion of the PutA protein in the presence and absence of proline demonstrated that the persistence of a 119-kDa protein fragment was characteristic of the reduced protein. Identical digestion patterns were obtained from the apoprotein in the presence and absence of proline. The quantity of the 119-kDa fragment produced varied with proline concentration, yielding a midpoint of 0.056 mM proline. The fraction of PutA protein associated with membrane vesicles was also a function of proline concentration, yielding a titration midpoint of 0.10 mM proline. Membrane binding was thus coincident with both flavin reduction and a change in protein conformation.