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

Abstract

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.

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

“…ALDHs share a similar two-step mechanism involving an acylation step and a deacylation step. The acylation step initiates the formation of a covalent hemithioacetal intermediate via the nucleophilic attack of the catalytic cysteine (Cys302 for human liver ALDH) on the aldehyde substrate followed by the hydride transfer that leads to the formation of a thioacylenzyme intermediate and NAD(P)H [15,38]. Cys289 of BlALDH could be the active-site residue since it corresponds to Cys302 of human liver ALDH and is conserved in the aligned enzymes (Fig.…”

“…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].…”

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae

“…Further, Fig. 1 (Dickinson & Haywood, 1986). Ofcourse, ifother kinetically significant species are involved in the dissociation sequence from NADH E-acyl than are indicated in Scheme 1, there would K+ be other common steps for dehydrogenase and NADHactivated esterase reactions and other potential ratelimiting steps.…”

“…dehydrogenase (Dickinson & Haywood, 1986) intramolecular event before it. These conclusions indicate that the product release sequence is more complex than shown in Scheme 1.…”

“…3-fold) by Mg2" (Dickinson & Haywood, 1986). The step so influenced appears to be hydrolysis of the acyl-enzyme species, and this process occurs more rapidly with NADH NADH E-acyl than with E-acyl.…”