Investigation of the effects of the N-terminal portion of aldehyde dehydrogenase on solubility, stability, and mitochondria import and the dominance of the Oriental K487 subunit over the E487 subunit in heterotetrameric mitochondrial aldehyde dehydrogenase

Jianzhong Zhou, Purdue University

Abstract

Two major isozymes of aldehyde dehydrogenase, cytosolic ALDH1 and mitochondrial ALDH2, have virtually identical tertiary structures and share 70% protein sequence identity except for the first 21 amino acids (14%). Chimeras were made by exchanging the 21 residues, which are on the surface of the protein, between the two isozymes. It was found that the chimera with a majority of residues from ALDH1 was soluble and active while the ALDH2 chimera, containing the 21 residues from ALDH1, was insoluble. After restoring two interactions between these 21 residues and the remaining ALDH2 protein, a soluble protein was obtained and the activity recovered. The ALDH1 chimera was less stable than its parent but was still more stable than ALDH2. The import of precursor ALDH1 was much poorer than that of ALDH2. The reason was that ALDH1 folded faster than ALDH2 and that the in vitro synthesized ALDH1 could assemble to the tetrameric form while ALDH2 remained a monomer. Exchanging the N-terminal portion altered import by changing the properties of folding, assembly, and stability. An Oriental variant of ALDH2 that has a lysine rather than a glutamate at position 487 has low activity and a high Km for NAD+, compared to the non-Oriental ALDH2. The heterotetrameric ALDH2, containing E487 and K487 subunits, had lower activity than expected. It appeared that the K487 subunits altered the activity of the E487 subunits. Surface residues were changed so that various heterotetrameric forms of the enzyme could be investigated. By measuring specific activity, burst formation of NADH, and stimulation of esterase activity by NAD+ with the individual homogenous recombinantly expressed heterotetrameric ALDHs, we concluded that a K487 subunit could affect an E487 subunit in the same dimer pair but not in the other dimer pair. In heterotetrameric ALDH the E-E dimer pair was active while the E-K dimer behaved like a K-K dimer. The halfof-the-site reactivity apparently resulted from there being one active site in each dimer pair of the tetrameric enzyme and not one fully active dimer and one inactive dimer in the tetramer.

Degree

Ph.D.

Advisors

Weiner, Purdue University.

Subject Area

Biochemistry

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