Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD⁺ as a coenzyme. Porcine kidney BADH (pkBADH) follows a bi‐bi ordered mechanism in which NAD⁺ binds to the enzyme before the aldehyde. Previous studies showed that NAD⁺ induces complex and unusual conformational changes on pkBADH and that potassium is required to maintain its quaternary structure. The aim of this work was to analyze the structural changes in pkBADH caused by NAD⁺ binding and the role played by potassium in those changes. The pkBADH cDNA was cloned and overexpressed in Escherichia coli, and the protein was purified by affinity chromatography using a chitin matrix. The pkBADH/NAD⁺ interaction was analyzed by circular dichroism (CD) and by isothermal titration calorimetry (ITC) by titrating the enzyme with NAD⁺. The cDNA has an open reading frame of 1485 bp and encodes a protein of 494 amino acids, with a predicted molecular mass of 53.9 kDa. CD data showed that the binding of NAD⁺ to the enzyme caused changes in its secondary structure, whereas the presence of K⁺ helps maintain its α‐helix content. K⁺ increased the thermal stability of the pkBADH‐NAD⁺ complex by 5.3°C. ITC data showed that NAD⁺ binding occurs with different association constants for each active site between 37.5 and 8.6 μM. All the results support previous data in which the enzyme incubation with NAD⁺ provoked changes in reactivity, which is an indication of slow conformational rearrangements of the active site.

中文翻译：

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重组甜菜碱醛脱氢酶活性位点的异质性受钾调节。

甜菜碱醛脱氢酶 (BADH EC 1.2.1.8) 使用 NAD ⁺作为辅酶催化甜菜碱醛不可逆氧化为甘氨酸甜菜碱。猪肾 BADH (pkBADH) 遵循双双有序机制，其中 NAD ⁺在醛之前与酶结合。以前的研究表明，NAD ⁺诱导 pkBADH 上复杂且不寻常的构象变化，并且需要钾来维持其四级结构。这项工作的目的是分析由 NAD ⁺结合引起的 pkBADH 结构变化以及钾在这些变化中所起的作用。pkBADH cDNA 在大肠杆菌中被克隆和过表达，并使用几丁质基质通过亲和层析纯化蛋白质。pkBADH/NAD ⁺相互作用通过圆二色性 (CD) 和等温滴定量热法 (ITC) 通过用 NAD ⁺滴定酶进行分析。该 cDNA 的开放阅读框为 1485 bp，编码 494 个氨基酸的蛋白质，预测分子量为 53.9 kDa。CD 数据显示 NAD ⁺与酶的结合导致其二级结构发生变化，而 K ⁺的存在有助于维持其 α-螺旋含量。K ⁺将 pkBADH-NAD ⁺复合物的热稳定性提高了5.3°C。ITC 数据显示 NAD ⁺每个活性位点的结合常数在 37.5 和 8.6 μM 之间具有不同的结合常数。所有结果都支持先前的数据，其中酶与 NAD ^{+ 的}温育引起反应性变化，这是活性位点构象重排缓慢的迹象。