U.S. flag

An official website of the United States government

Dot gov

Official websites use .gov
A .gov website belongs to an official government organization in the United States.


Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.


Main content area

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

Thomas P. Gonnella, Jennifer M. Keating, Jessica A. Kjemhus, Matthew J. Picklo, Joseph P. Biggane
Chemico-biological interactions 2013 v.202 no.1-3 pp. 85-90
NAD (coenzyme), isomerization, humans, fluorescence, binding properties, aldehyde dehydrogenase, cations, dissociation, enzymatic reactions, magnesium, spectroscopy, oxidation, enzyme activity, catalytic activity, aldehydes, carboxylic acids
Aldehyde dehydrogenase 1 (ALDH1A1) catalyzes the oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) 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 (?) of free NADH in solution (?=0.4 ns) and two enzyme-bound NADH states (?=2.0 ns and ?=7.7 ns). We used this technique to investigate the effects of Mg(2+) 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 24 ?M to 1 ?M for Mg(2+) ion concentrations of 0-6000 ?M, respectively. The rate constants for dissociation of the enzyme-NADH complex ranged from 0.03 s(-1) (6000 ?M Mg(2+)) to 0.30s(-1) (0 ?M Mg(2+)) 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 Mg(2+) ion concentration was increased, there was a consistent decrease of the enzyme catalytic turnover from 0.31 s(-1) (0 ?M Mg(2+)) to 0.050 s(-1) (6000 ?M Mg(2+)) 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 6000 ?M Mg(2+). This shift in conformational population at higher Mg(2+) 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.