|GONNELLA, THOMAS - Mayville State University|
|LEEDAHL, TRAVIS - Mayville State University|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 6/2/2010
Publication Date: 1/27/2011
Citation: Gonnella, T.P., Leedahl, T.S., Picklo, M.J. 2011. NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2. Enzymology and Molecular Biology of Carbonyl Metabolism 15th International Meeting. PMID: 21276780.
Technical Abstract: ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of ALDH2-NADH interaction. By using time-resolved fluorescence spectroscopy, we have resolved free NADH (tau = 0.4 ns) and bound NADH (tau = 6.0 ns). We used this technique to investigate the effects of Mg2+ on the ALDH2-NADH binding characteristics and enzyme catalysis. We assessed the ALDH2-NADH dissociation constant (Kd) by sequential addition of NADH to mixtures of recombinant rat ALDH2 with varying Mg2+ ion concentrations and monitoring the levels of free versus bound NADH. From the resolved free and bound NADH fluorescence signatures, the Kd for NADH with ALDH2 ranged from 334 uM to 10 uM for Mg2+ ion concentrations of 2 uM to 6000 uM, respectively. We determined the effects of Mg 2+ ion on the NADH dissociation and association rates by displacing NADH from ALDH2 with the addition of excess NAD+ and resolving the real-time NADH fluorescence signal. The dissociation rate of NADH ranged from 0.4 s-1 (6000 uM Mg2+) to 8.3 s-1 (0 uM Mg2+). From the determined Kd values and NADH dissociation rates, the calculated NADH association/re-association rates were fairly consistent ~0.04 s-1 over the entire Mg2+ ion concentration range. These data demonstrate that Mg 2+ slows the release of NADH from the enzyme rather than promotes its re-association. We applied our NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Reactions were performed with ALDH2, saturating levels of NAD+ and increasing levels of Mg2+ followed by addition of propanal to initiate the reaction. Our fluorescence lifetime analysis confirmed the biphasic behavior of the enzyme activity as a function of Mg2+ concentration. Importantly, we observed no pre-steady state burst of NADH formation in either the presence or absence of Mg2+, and our results indicate that previously reported burst activity is an artifact of the increased NADH fluorescence efficiency of the ALDH2-NADH complex that follows the hydride transfer step. 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 (tau = 11.2 ns).