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ARS Home » Plains Area » Grand Forks, North Dakota » Grand Forks Human Nutrition Research Center » Dietary Prevention of Obesity-related Disease Research » Research » Publications at this Location » Publication #285326

Title: Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

item GONNELLA, THOMAS - Mayville State University
item KEATING, JENNIFER - Mayville State University
item KJEMHUS, JESSICA - Mayville State University
item Picklo, Matthew
item BIGGANE, JOSEPH - Mayville State University

Submitted to: Chemico Biological Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2012
Publication Date: 2/25/2013
Publication URL:
Citation: Gonnella, T.P., Keating, J.M., Kjemhus, J.A., Picklo, M.J., Biggane, J.P. 2013. Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1. Chemico Biological Interactions. 202(1-3):85-90.

Interpretive Summary: Magnesium is an essential dietary component that modulates several cellular processes. In this work, we examined the role of magnesium in the regulation of the activity of the cellular defense enzymes aldehyde dehydrogenases (ALDHs) that are found in the liver. Using ultra-rapid fluorescence measurements, we were able to monitor the different states of NADH during its binding to and release from the ALDH1 enzyme. Our results demonstrate that magnesium ions decrease the activity of the ALDH1 enzyme in a concentration-dependent manner through inhibiting the release of the enzyme product NADH. These data provide insight into how dietary components regulate cellular protection mechanisms.

Technical Abstract: Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (t) of free NADH in solution (t = 0.4 ns) and two enzyme-bound NADH states (t = 2.0 ns and t = 7.7 ns). We used this technique to investigate the effects of Mg2+ on the ALDH1-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD values for both NADH conformations in ALDH1 ranged from about 25 µM to 1 µM for Mg2+ ion concentrations of 0 µM to 6000 µM, respectively. The rate constants for dissociation of the enzyme-NADH complex ranged from 0.03 s-1 (6000 µM Mg2+) to 0.30 s-1 (0 µM Mg2+) as determined by addition of excess NAD+ to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed consistent decrease of the enzyme activity as a function of Mg2+ concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct shift in conformation population from predominantly one ALDH1-NADH complex (t = 2.0 ns) at in the absence of magnesium ion to a nearly equal mixture of ALDH1-NADH complexes at high magnesium concentrations. This shift in population at higher Mg2+ concentrations and lower enzyme activity may be due to longer residence time of the NADH inside the ALDH1 pocket. The results from monitoring enzyme catalysis in the absence of magnesium suggests that the ALDH1-NADH complex with the shorter fluorescence lifetime is the form initially produced and the complex with the longer fluorescence lifetime is produced through isomerization of the former.