Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD⁺ as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD⁺ with different affinities at each active site and that the binding is K⁺ dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD⁺ and the role played by K⁺. Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K⁺ and titrating the enzyme with varying concentrations of NAD⁺. Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K⁺ was increased. Changes in the exposure of tryptophan located near the NAD⁺ binding site were found when the enzyme was titrated with NAD⁺ in the presence of potassium. Fluorescence data analysis showed that the K⁺ presence promoted static quenching that facilitated the pkBADH–NAD⁺ complex formation. DC data analysis showed that binding of K⁺ to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K⁺, respectively. The presence of K⁺ during NAD⁺ binding to pkBADH increased the thermal stability of the complex. These results indicated that K⁺ facilitated the pkBADH–NAD⁺ complex formation and suggested that K⁺ caused small changes in secondary and tertiary structures that could influence the active site conformation.

中文翻译：

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辅酶结合依赖钾的甜菜碱醛脱氢酶的光谱分析

甘氨酸甜菜碱是哺乳动物肾细胞中合成和积累的主要渗透剂。甘氨酸甜菜碱合成由甜菜碱醛脱氢酶 (BADH) 催化，使用 NAD ⁺作为辅酶。先前的研究表明，猪肾甜菜碱醛脱氢酶 (pkBADH)在每个活性位点以不同的亲和力结合 NAD ⁺并且这种结合是 K ⁺依赖性的。这项工作的目的是分析由不同浓度的 NAD ⁺引起的 pkBADH 二级和三级结构的变化以及 K ^{+ 所}起的作用。在固定可变浓度的 K ^{+ 下}进行本征荧光研究并用不同浓度的 NAD ⁺滴定酶。荧光分析表明，随着 K ⁺浓度的增加，最大发射向红色移动。在存在钾的情况下用 NAD ⁺滴定酶时，发现位于 NAD ⁺结合位点附近的色氨酸暴露发生变化。荧光数据分析表明，K ^{+ 的}存在促进了静态猝灭，从而促进了 pkBADH-NAD ⁺复合物的形成。DC数据分析表明，在25 mM和100 mM K ⁺存在下，K ⁺与酶的结合导致α-螺旋含量发生4%和12%的变化， 分别。NAD ⁺与 pkBADH 结合期间K ⁺的存在增加了复合物的热稳定性。这些结果表明 K ⁺促进了 pkBADH-NAD ⁺复合物的形成，并表明 K ⁺引起二级和三级结构的微小变化，这可能会影响活性位点构象。