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

César Muñoz-Bacasehua, Jesus A Rosas-Rodríguez, Alexis Alonso López-Zavala, Elisa M Valenzuela-Soto

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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.

Original languageEnglish
JournalLuminescence : the journal of biological and chemical luminescence
StateE-pub ahead of print - 2 Jul 2021

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