Role of potassium levels in pkBADH heterogeneity of NAD⁺ binding site

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; K_D 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 TΔS_b 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 (T_m)_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.