Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent of 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 coenzyme. Previous studies showed that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD+ with different affinity 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 resulted 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 shows a shift of the maximum emission towards red as the concentration of K+ increases. 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 K+ presence promoted a static quenching that facilitates pkBADH-NAD+ complex formation. DC data analysis showed that binding of K+ to the enzyme causes 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 indicate that K+ facilitates the pkBADH-NAD+ complex formation and suggest that K+ causes small changes in secondary and tertiary structure that can influence the active site conformation.