Heterotetramers of human liver mitochondrial (class 2) aldehyde dehydrogenase expressed in Escherichia coli. A model to study the heterotetramers expected to be found in Oriental people.
About 50% of the Oriental population have less liver mitochondrial aldehyde dehydrogenase (ALDH2) activity than do other people. It was found that they possessed an enzyme with a lysine at position 487 (E487K) instead of glutamate (Glu487). We previously found that the Km for NAD of recombinant human and rat E487K enzymes increased more than 150-fold (Farrés, J., Wang X., Takahashi, K., Cunningham, S. J. , Wang, T.T., and Weiner, H (1994) J. Biol. Chem. 269, 13854-13860). Many aldehyde dehydrogenase-deficient people were found to be heterozygous when genotyped for ALDH2. In this study liver tissue from heterozygous people was analyzed and found to possess mRNAs for both the glutamate and the lysine subunits. Western blot analysis showed that the glutamate subunit was present. The cDNAs for Glu487 and E487K were coexpressed on one plasmid in Escherichia coli, and the enzyme forms were separated from each other by isoelectric focusing to show that heterotetramers were formed. Only one Km value for NAD could be measured with the purified heterotetrameric enzyme that possessed just 16-18% activity of the glutamate homotetrameric enzyme. The E487K homotetramers had 8% specific activity of the Glu487 enzyme. There was no pre-steady state burst of NADH formation with the heterotetramer, a property found with the glutamate enzyme. Similar results were found for the coexpressed rat liver enzyme, except that a higher specific activity, 48%, was obtained. Thus, we conclude that presence of the lysine subunit altered the activity of the glutamate subunit in the heterotetramer to make it function more like an E487K enzyme.
Wang
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X
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA. weiner@biochem.purdue.edu
Sheikh
S
S
Saigal
D
D
Robinson
L
L
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D044466:Asian People; D004926:Escherichia coli; D018698:Glutamic Acid; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D018698:Glutamic Acid; D000444:Aldehyde Dehydrogenase