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.
|Number of pages||10|
|State||Published - Nov 2021|
Bibliographical noteFunding Information:
César Muñoz-Bacasehua gratefully acknowledges a scholarship from CONACyT for Ph.D. studies. Authors are grateful to G. Garza-Ramos for providing facilities and constant support to carry out CD spectra.
© 2021 John Wiley & Sons, Ltd.
- betaine aldehyde dehydrogenase
- coenzyme binding
- negative cooperativity
- protein structure model