Involvement of glutamate 399 and lysine 192 in the mechanism of human liver mitochondrial aldehyde dehydrogenase.
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.
Ni
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Sheikh
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S
Weiner
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H
D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000447:Aldehydes / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D000447:Aldehydes; D018698:Glutamic Acid; C005556:propionaldehyde; C004656:chloroacetaldehyde; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000079:Acetaldehyde; D008274:Magnesium; D008239:Lysine