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 Escherichiae 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 pK(a) 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 V(max) increased, as did the K(m) for NAD+, while the K(m) for propionaldehyde decreased. H235A had the same pI as the native enzyme but the V(max) 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 esidue at position 235 may be required for the newly synthesized peptide to fold and/or assemble into the native conformation of ALDH. © 1993 Academic Press, Inc.