Betaine aldehyde dehydrogenase (BADH) catalyzes the oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Studies in porcine kidney BADH (pkBADH) suggested that the enzyme exhibits heterogeneity of active sites and undergoes potassium-induced conformational changes. This study aimed to analyze if potassium concentration plays a role in the heterogeneity of pkBADH active sites through changes in NAD+ affinity constants, in its secondary structure content and stability. The enzyme was titrated with NAD+ 1 mM at fixed-variable KCl concentration, and the interaction measured by Isothermal Titration Calorimetry (ITC) and Circular Dichroism (CD). ITC data showed that K+ increased the first active site affinity in a manner dependent on its concentration; KD values to the first site were 14.4, 13.1, and 10.4 μM, at 25, 50, and 75 mM KCl. ΔG values showed that the coenzyme binding is a spontaneous reaction without changes between active sites or depending on KCl concentration. ΔH and TΔSb values showed that NAD+ binding to the active site is an endothermic process and is carried out at the expense of changes in entropy. α-Helix content increased as KCl increased, enzyme (Tm)app values were 2.6 °C and 3.3 °C higher at 20 mM and 200 mM K+. PkBADH molecular model showed three different interaction K+ sites. Results suggested K+ can interact with pkBADH and cause changes in the secondary structure, it provokes changes in the enzyme affinity by the coenzyme, and in the thermostability.

中文翻译：

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钾水平在NAD +结合位点pkBADH异质性中的作用。

甜菜碱醛脱氢酶（BADH）使用NAD +作为辅酶催化甜菜碱醛氧化为甘氨酸甜菜碱。猪肾脏BADH（pkBADH）的研究表明，该酶表现出活性位点的异质性并经历钾诱导的构象变化。这项研究旨在分析钾浓度是否通过改变NAD +亲和常数，二级结构含量和稳定性来在pkBADH活性位点的异质性中起作用。酶以固定可变的KCl浓度用NAD + 1 mM滴定，并通过等温滴定热量法（ITC）和圆二色谱（CD）测定相互作用。ITC数据显示，K +以依赖于其浓度的方式增加了第一个活性位点的亲和力。到第一个位点的KD值为25、50、14.4、13.1和10.4μM，和75 mM KCl。ΔG值表明，辅酶结合是自发反应，活性位点之间没有变化，也没有KCl浓度的影响。ΔH和TΔSb值表明NAD +与活性位点的结合是一个吸热过程，并且是以熵变化为代价进行的。随着KCl的增加，α-螺旋含量增加，在20 mM和200 mM K +时，酶（Tm）app值分别升高2.6°C和3.3°C。PkBADH分子模型显示了三个不同的相互作用K +位点。结果表明，K +可以与pkBADH相互作用并引起二级结构的变化，它引起辅酶的酶亲和力和热稳定性的变化。ΔH和TΔSb值表明NAD +与活性位点的结合是一个吸热过程，并且以熵变为代价进行。随着KCl的增加，α-螺旋含量增加，在20 mM和200 mM K +时，酶（Tm）app值分别升高2.6°C和3.3°C。PkBADH分子模型显示了三个不同的相互作用K +位点。结果表明，K +可以与pkBADH相互作用并引起二级结构的变化，它引起辅酶的酶亲和力和热稳定性的变化。ΔH和TΔSb值表明NAD +与活性位点的结合是一个吸热过程，并且以熵变为代价进行。随着KCl的增加，α-螺旋含量增加，在20 mM和200 mM K +时，酶（Tm）app值分别升高2.6°C和3.3°C。PkBADH分子模型显示了三个不同的相互作用K +位点。结果表明，K +可以与pkBADH相互作用并引起二级结构的变化，它引起辅酶的酶亲和力和热稳定性的变化。