NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2–NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ = 0.4 ns) and bound NADH (τ = 6.0 ns). We used this technique to investigate the effects of Mg2+ on the ALDH2–NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD for NADH with ALDH2 ranged from 468 μM to 12 μM for Mg2+ ion concentrations of 20 to 6000 μM, respectively. The rate constant for dissociation of the enzyme–NADH complex ranged from 0.4 s−1 (6000 μM Mg2+) to 8.3 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. The apparent NADH association/re-association rate constants were approximately 0.04 μM−1 s−1 over the entire Mg2+ ion concentration range and demonstrate that Mg2+ ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior 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 fluorescence signatures from multiple ALDH2–NADH complexes corresponding to free NADH, enzyme–bound NADH, and, potentially, an abortive NADH–enzyme–propanal complex (τ = 11.2 ns).