Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase

Abstract

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme

“…The occurrence of transients on different time scales in the spectrophotometric and fluorescence records has already been reported for this enzyme (Hart & Dickinson, 1982a) under conditions similar to those of Fig. 2.…”

“…The apparatus used was that described by Hart & Dickinson (1982a). The performance of the apparatus was tested as described earlier (Dickinson, 1985).…”

Gene Cloning and Biochemical Characterization of a NAD(P)+-Dependent Aldehyde Dehydrogenase from Bacillus licheniformis

“…Based on the sequences, the enzymes were placed into different families or classes [2,12], including phenylacetaldehyde dehydrogenase, lactaldehyde dehydrogenase, bacterial ALDH, human ALDH (class 1, ALDH1; class 2, ALDH2; class 3, ALDH3), and yeast ALDH (class 1, yALDH1; class 5, yALDH5; class 2, yALDH2). Though many of these enzymes have been characterized with respect to substrate specificity, perhaps the best-studied ones are the mammalian ALDH from liver cytosol (class 1) [13][14][15], liver mitochondria (class 2) [16][17][18][19][20], stomach cytosol (class 3) [21][22][23][24], and the bacteria ALDH from Escherichia coli [25], Vibrio cholerae [26], and Alcaligenes eutrophus [27].…”

Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases

“…Though many of the enzymes have been characterized with respect to substrate specificity, mechanistic work has been done with just a few. Perhaps the best‐studied ones are the mammalian enzymes from liver cytosol (class 1) (Hart and Dickinson 1982; Vallari and Pietruszko 1984; Dickinson and Haywood 1986), liver mitochondria (class 2) (Feldman and Weiner 1972a, b; Sidhu et al 1975; Zheng et al 1993; Ni et al 1997; Sheikh et al 1997), and dimeric liver and stomach cytosol (class 3) (Hsu et al 1992; Mann and Weiner 1999; Perozich et al 2000; Hempel et al 2001;). Some generalizations about the enzymes have been presented based on properties of the enzymes found in these three classes.…”