Role of the highly conserved histidine residues in rat liver mitochondrial aldehyde dehydrogenase as studied by site-directed mutagenesis.
One histidine residue (H235) is conserved in all known aldehyde dehydrogenases (ALDH), from Escherichia coli to human, except for those from P. oleovorans and rat hepatoma. Kinetic studies with horse liver mitochondrial ALDH indicated that a group with a pKa of 7 may be involved in the active site. Using site-directed mutagenesis, the four conserved histidine residues of the six histidines in rat liver mitochondrial ALDH were converted to alanines. Only modification of H235 and H29 caused alterations in properties of the enzyme. H29A had a decreased pI suggesting that this residue may normally be protonated. Its Vmax increased, as did the Km for NAD+, while the Km for propionaldehyde decreased. H235A had the same pI as the native enzyme but the Vmax decreased by 50%. Like native enzyme, H235A was active in Hepes and Mops buffer as well as in phosphate buffer. Purified H235A was thermally less stable than was native enzyme. H235 was also changed to F, Y, E, K, and Q. All of these substitutions resulted in the formation of insoluble aggregates or inclusion bodies when they were expressed in E. coli. It appears then that the highly conserved histidine residues may not be functioning as a general base in the deacylation step as we originally suggested. Instead, both H29 and H235 may be of structural importance and the presence of a histidine residue at position 235 may be required for the newly synthesized peptide to fold and/or assemble into the native conformation of ALDH.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
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D000409:Alanine / Q000737:chemistry; D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D002384:Catalysis; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship
D002021:Buffers; D011994:Recombinant Proteins; D006639:Histidine; D000444:Aldehyde Dehydrogenase; D000409:Alanine