title: Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1.
abstract: 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 (τ) of free NADH in solution (τ=0.4 ns) and two enzyme-bound NADH states (τ=2.0 ns and τ=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 μM to 1 μM for Mg(2+) ion concentrations of 0-6000 μM, respectively. The rate constants for dissociation of the enzyme-NADH complex ranged from 0.03 s(-1) (6000 μM Mg(2+)) to 0.30s(-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. 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 μM Mg(2+)) to 0.050 s(-1) (6000 μM 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 μM 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.
authors.affiliation: Division of Science and Mathematics, Mayville State University, Mayville, ND 58257, USA. tom.gonnella@mayvillestate.edu
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Gonnella
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Keating
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Kjemhus
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Picklo
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Biggane
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000080924:Aldehyde Dehydrogenase 1 Family; D000447:Aldehydes / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D050697:Retinal Dehydrogenase; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D000447:Aldehydes; D002413:Cations, Divalent; D007527:Isoenzymes; D009243:NAD; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; C018475:butyraldehyde; D008274:Magnesium