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

Authors: Gonnella, TP  Leedahl, TS  Karlstad, JP  Picklo, MJ 
Citation: Gonnella TP, etal., Chem Biol Interact. 2011 May 30;191(1-3):147-52. doi: 10.1016/j.cbi.2011.01.023. Epub 2011 Jan 27.
Pubmed: (View Article at PubMed) PMID:21276780
DOI: Full-text: DOI:10.1016/j.cbi.2011.01.023

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 (tau) of free NADH (tau=0.4 ns) and bound NADH (tau=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 muM to 12 muM for Mg(2+) ion concentrations of 20 to 6000 muM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4s(-1) (6000 muM Mg(2+)) to 8.3s(-1) (0 muM 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 muM(-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 (tau=11.2 ns).


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RGD Object Information
RGD ID: 7241587
Created: 2013-03-12
Species: All species
Last Modified: 2013-03-12
Status: ACTIVE


RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.