Basis for half-of-the-site reactivity and the dominance of the K487 oriental subunit over the E487 subunit in heterotetrameric human liver mitochondrial aldehyde dehydrogenase.
Human liver mitochondrial aldehyde dehydrogenase is a tetrameric enzyme composed of 4 identical 500 amino acid containing subunits arranged such that the protein is a dimer of dimers. No kinetic evidence for subunit interactions has been reported. However, the enzyme exhibits half-of-the-site reactivity in that there is a pre-steady-state burst of 2 mol of NADH per mole of enzyme. A variant of the enzyme, found in Asian people, contains a lysine rather than a glutamate at position 487. This enzyme has a high K(M) for NAD(+) and a low specific activity. In heterotetramers composed of both subunit types, it appeared that the lysine-containing subunit was dominant over the glutamate-containing subunits. To allow for the separation of various heterotetrameric forms of the enzyme, surface residues were changed. Each of the five possible tetrameric forms of the modified enzyme was isolated and characterized with respect to steady-state kinetics and pre-steady-state burst magnitudes. The data best fit a model where in each dimer pair there is one functioning and one nonfunctioning subunit. Further, the lysine subunit affects the properties only of its dimer partner. Residue 487 is located at the dimer interface, and the glutamate forms salt bonds with two arginine residues. One is to Arg(264) in the same subunit; the other is to Arg(475) located in the other subunit. Most likely the presence of a lysine affects these salt bonds so the lysine subunit can cause the other subunit to become essentially nonfunctional.
Zhou
J
J
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D044466:Asian People / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D009243:NAD; D018698:Glutamic Acid; D000444:Aldehyde Dehydrogenase; D008239:Lysine