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

• Published in: Chemico-biological interactions 2013-02, Vol.202 (1-3), p.85-90
• Main Authors: Gonnella, Thomas P., Keating, Jennifer M., Kjemhus, Jessica A., Picklo, Matthew J., Biggane, Joseph P.
• Format: Article
• Language: English
• Subjects: Aldehyde dehydrogenase; Aldehyde Dehydrogenase - chemistry; Aldehyde Dehydrogenase - metabolism; Aldehydes - metabolism; Binding Sites; Catalysis; Cations, Divalent - chemistry; Cations, Divalent - metabolism; Fluorescence; Humans; Isoenzymes - metabolism; Magnesium - chemistry; Magnesium - metabolism; NAD - chemistry; NAD - metabolism; NADH; Oxidation-Reduction; Protein Binding; Spectrometry, Fluorescence - methods
• ISSN: 0009-2797; 1872-7786
• DOI: 10.1016/j.cbi.2012.12.008

Aldehyde dehydrogenase 1 (ALDH1A1) catalyzes the oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions decrease ALDH1 activity in part by increasing NADH binding affinity to the enzyme. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH in solution (τ=0.4ns) and two enzyme-bound NADH states (τ=2.0ns and τ=7.7ns). We used this technique to investigate the effects of Mg2+ ions on the ALDH1A1–NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD values for both NADH conformations in ALDH1A1 ranged from about 24μM to 1μM for Mg2+ ion concentrations of 0–6000μM, respectively. The rate constants for dissociation of the enzyme–NADH complex ranged from 0.03s−1 (6000μM Mg2+) to 0.30s−1 (0μM Mg2+) as determined by addition of excess NAD+ to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. During the initial reaction of enzyme with NAD+ and butyraldehyde, there was an immediate rise in the NADH fluorescence, due to the formation of bound NADH complexes, with a constant steady-state rate of production of free NADH. As the Mg2+ ion concentration was increased, there was a consistent decrease of the enzyme catalytic turnover from 0.31s−1 (0μM Mg2+) to 0.050s−1 (6000μM Mg2+) and a distinct shift in steady-state conformational population from one that favors the ALDH1–NADH complex with the shorter fluorescence lifetime (33% excess) in the absence of magnesium ion to one that favors the ALDH1–NADH complex with the longer fluorescence lifetime (13% excess) at 6000μM Mg2+. This shift in conformational population at higher Mg2+ ion concentrations and to lower enzyme activity may be due to longer residence time of the NADH in 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.