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

Abstract

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) 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 Mg(2+) on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the K(D) for NADH with ALDH2 ranged from 468 μM to 12 μM for Mg(2+) ion concentrations of 20 to 6000 μM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4s(-1) (6000 μM Mg(2+)) to 8.3s(-1) (0 μM Mg(2+)) 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 Mg(2+) ion concentration range and demonstrate that Mg(2+) 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 Mg(2+) 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).

Figure 1. Schematic of the ALDH2 catalytic mechanism

Figure 2. An example wavelength-time matrix (WTM) of the NADH-ALDH2 complex

The sample contained 1.1 µM NADH, 0.5 µM ALDH2, and 5.0 mM Mg²⁺ in 40 mM HEPES buffer (pH 7.4). WTMs are used to resolve the fluorescent lifetime (τ) and emission spectrum of each contributing fluorophore.

Figure 3. Mg²⁺ alters ALDH2 activity in a complex manner

The effect of Mg²⁺ ion concentration on enzyme activity. Reaction mixtures contained 0.25 µM ALDH2 (tetramer), 1 mM NAD⁺, 100 µM propanal, and the indicated Mg²⁺ concentrations in 40 mM HEPES buffer, pH 7.4.

Figure 4. Time-resolved fluorescence analysis of ALDH2 activity

(A) The fluorescence waveforms collected during enzymatic catalysis are used to determine the changes in concentration of free and bound forms of NADH. In addition to the fluorescence from the free NADH (τ = 0.4 ns) and ALDH2-NADH complex (τ = 6.0 ns) observed in the K_D studies, a third fluorescent contributor (τ = 11.2 ns) is resolved in the presence of Mg²⁺. The reaction mixture contained 0.25 µM ALDH2, 1 mM NAD⁺, 100 µM propanal, and 6000 µM Mg²⁺. (B) The effect of Mg²⁺ ion concentration on relative fluorescence intensities reached at steady state conditions from the ALDH2-NADH complex and third fluorescent contributor.

Figure 4. Time-resolved fluorescence analysis of ALDH2 activity

(A) The fluorescence waveforms collected during enzymatic catalysis are used to determine the changes in concentration of free and bound forms of NADH. In addition to the fluorescence from the free NADH (τ = 0.4 ns) and ALDH2-NADH complex (τ = 6.0 ns) observed in the K_D studies, a third fluorescent contributor (τ = 11.2 ns) is resolved in the presence of Mg²⁺. The reaction mixture contained 0.25 µM ALDH2, 1 mM NAD⁺, 100 µM propanal, and 6000 µM Mg²⁺. (B) The effect of Mg²⁺ ion concentration on relative fluorescence intensities reached at steady state conditions from the ALDH2-NADH complex and third fluorescent contributor.

Figure 5. Resolved spectral and temporal contributions from WTM of a sample containing 2.9 µM NADH, 0.25 µM ALDH2, and 6.0 mM Mg²⁺ in 40 mM HEPES buffer (pH 7.4)

(A) The resolved fluorescence spectra illustrates that the third contributor has an identical fluorescent spectrum to that of the ALDH2-NADH complex. (B) The resolved temporal contributions provide a measure of the relative amounts and the fluorescence lifetime of each contributor.

Figure 5. Resolved spectral and temporal contributions from WTM of a sample containing 2.9 µM NADH, 0.25 µM ALDH2, and 6.0 mM Mg²⁺ in 40 mM HEPES buffer (pH 7.4)

(A) The resolved fluorescence spectra illustrates that the third contributor has an identical fluorescent spectrum to that of the ALDH2-NADH complex. (B) The resolved temporal contributions provide a measure of the relative amounts and the fluorescence lifetime of each contributor.

Figure 6. Proposed schematic of ALDH2-NADH-propanal abortive ternary complex in the presence of Mg²⁺