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.[1]References
- Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis. Ho, K.K., Hurley, T.D., Weiner, H. Biochemistry (2006) [Pubmed]
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