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 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 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) 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.