Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent on potassium

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. Potassium caused changes in the exposure tryptophans located near the NAD+ binding site when the enzyme was titrated with NAD+. Potassium presence promoted static quenching that facilitated pkBADH–NAD+ complex formation. Potassium presence during NAD+ binding caused changes in the pkBADH α‐helix content and increased complex thermal stability.