Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis

Kwok Ki Ho, Thomas D. Hurley, Henry Weiner

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 Mg2+ 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 Mg2+ ion activation. An ALDH1 T244S mutant was recombinantly expressed and was used for mechanistic studies. Mg2+ 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, Mg2+ 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.

Original languageEnglish (US)
Pages (from-to)9445-9453
Number of pages9
JournalBiochemistry
Volume45
Issue number31
DOIs
StatePublished - Aug 8 2006

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Mutagenesis
Aldehyde Dehydrogenase
NAD
Ions
Niacinamide
Acetaldehyde
Coenzymes
Enzymes
Amino Acid Substitution
Point Mutation
Hydrolysis
Assays
Sheep
Screening
Substitution reactions
Metals
Chemical activation
aldehyde dehydrogenase 1
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis. / Ho, Kwok Ki; Hurley, Thomas D.; Weiner, Henry.

In: Biochemistry, Vol. 45, No. 31, 08.08.2006, p. 9445-9453.

Research output: Contribution to journalArticle

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