Abstract
Mutation to the conserved Glu399 or Lys192 caused the rate-limiting step of human liver mitochondrial aldehyde dehydrogenase (ALDH2) to change from deacylation to hydride transfer (Sheikh, S., Ni, L., Hurley, T. D., and Weiner, H. (1997) J. Biol. Chem. 272, 18817-18822). Here we further investigated the role of these two NAD+-ribose-binding residues. The E399Q/K/H/D and K192Q mutants had lower dehydrogenase activity when compared with the native enzyme. No pre-steady state burst of NADH formation was found with the E399Q/K and K192Q enzymes when propionaldehyde was used as the substrate; furthermore, each mutant oxidized chloroacetaldehyde slower than propionaldehyde, and a primary isotope effect was observed for each mutant when [2H]acetaldehyde was used as a substrate. However, no isotope effect was observed for each mutant when alpha-[2H]benzaldehyde was the substrate. A pre-steady state burst of NADH formation was observed for the E399Q/K and K192Q mutants with benzaldehyde, and p-nitrobenzaldehyde was oxidized faster than benzaldehyde. Hence, when aromatic aldehydes were used as substrates, the rate-limiting step remained deacylation for all these mutants. The rate-limiting step remained deacylation for the E399H/D mutants when either aliphatic or aromatic aldehydes were used as substrates. The K192Q mutant displayed a change in substrate specificity, with aromatic aldehydes becoming better substrates than aliphatic aldehydes.
Highlights
The abbreviations used are: ALDH, aldehyde dehydrogenase; ALDH1, cytosolic aldehyde dehydrogenase; ALDH2, mitochondrial aldehyde dehydrogenase; ALDH3, microsomal aldehyde dehydrogenase; IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis; VH, the dehydrogenase activity of the enzyme when acetaldehyde or benzaldehyde was oxidized; VD, the dehydrogenase activity of the enzyme when [2H]acetaldehyde or a-[2H]benzaldehyde was oxidized
Expression and Purification of Native, E399Q/K/H/D, and K192Q/E Mutants of ALDH2 Enzymes—Recombinantly expressed native, Glu399, and Lys192 mutant forms of human liver mitochondrial ALDH were purified to homogeneity by established methods, as judged by SDS-PAGE followed by Coomassie Blue staining [2, 4, 8]
No primary isotope effect was found for the native ALDH2 enzyme with either a-[2H]benzaldehyde or [2H]acetaldehyde, consistent with hydride transfer not being the rate-limiting step for the native enzyme, and consistent with what we found for the horse liver mitochondrial ALDH [11, 12]
Summary
Materials—Benzaldehyde, p-nitrobenzaldehyde, and p-methoxybenzaldehyde were from Sigma; a-[2H]benzaldehyde (C6H5CDO) was from Cambridge Isotope Laboratories, Inc.; [2H]acetaldehyde (CD3CDO) was from Aldrich; IEF standards were from Bio-Rad; agarose for IEF and Pharmalytes were from Pharmacia Biotech Inc. Expression in E. coli and Purification of the Native and Mutant Enzymes—All mutated forms of the enzyme were expressed [2, 8] and purified as described previously [2, 4]. The protein concentration was determined as described previously [2, 4]. Fluorescence Assay for the Dehydrogenase Activity—The dehydrogenase activity assays were performed as described previously [2, 4, 9]. Determination of the Primary 2H Isotope Effect—Aldehyde dehydrogenase activity was determined at 25 °C in 100 mM sodium phosphate buffer (pH 7.4) in the presence of acetaldehyde and [2H]acetaldehyde, benzaldehyde, and a-[2H]benzaldehyde. Concentrations of NAD1 were 1–7 mM for the native and different mutant enzymes. The dehydrogenase activity assay was performed in the 100 mM sodium phosphate buffer (pH 7.4). Focused samples were detected by protein staining with Coomassie Blue [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
