Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis.
Random mutagenesis followed by a filter-based screening assay has been used to identify a mutant of human class 1 aldehyde dehydrogenase (ALDH1) that was no longer inhibited by Mg(2+) ions but was activated in their presence. Several mutants possessed double, triple, and quadruple amino acid substitutions with a total of seven different residues being altered, but each had a common T244S change. This point mutation proved to be responsible for the Mg(2+) ion activation. An ALDH1 T244S mutant was recombinantly expressed and was used for mechanistic studies. Mg(2+) ions have been shown to increase the rate of deacylation. Consistent with the rate-limiting step for ALDH1 being changed from coenzyme dissociation to deacylation was finding that chloroacetaldehyde was oxidized more rapidly than acetaldehyde. Furthermore, Mg(2+) ions only in the presence of NAD(H) increased the rate of hydrolysis of p-nitrophenyl acetate showing that the metal only affects the binary complex. Though the rate-limiting step for the T244S mutant was different from that of the native enzyme, the catalytic efficiency of the mutant was just 20% that of the native enzyme. The basis for the change in the rate-limiting step appears to be related to NAD(+) binding. Using the structure of a sheep class 1 ALDH, it was possible to deduce that the interaction between the side chain of T244 and its neighboring residues with the nicotinamide ring of NAD(+) were an essential determinant in the catalytic action of ALDH1.
Ho
Kwok Ki
KK
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Hurley
Thomas D
TD
Weiner
Henry
H
D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D002384:Catalysis; D002413:Cations, Divalent / Q000494:pharmacology; D019295:Computational Biology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000235:genetics; D004950:Esterases / Q000037:antagonists & inhibitors / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D016296:Mutagenesis; D009243:NAD / Q000737:chemistry; D009596:Nitrophenols / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D050697:Retinal Dehydrogenase; D012756:Sheep
D002413:Cations, Divalent; D007527:Isoenzymes; D009596:Nitrophenols; D009243:NAD; C008642:4-nitrophenyl acetate; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; D004950:Esterases; D008274:Magnesium