Bovine lens aldehyde dehydrogenase. Kinetics and mechanism.
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ABSTRACT:
SUBMITTER: Ting HH
PROVIDER: S-EPMC1152404 | biostudies-other | 1983 Nov
REPOSITORIES: biostudies-other
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Project description:Aldehyde reductase (aldose reductase) was purified to homogeneity (as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) from bovine lens by affinity chromatography on NADP+-Sepharose. The enzyme, a monomer of Mr about 40000, was active with a variety of alpha- hydroxyketones , including fructose. The minimum degree of the rate equation was 2:2 in the case of DL-glyceraldehyde, but linear kinetics were observed for glucose and NADPH over the concentration range studied. The enzyme largely followed a ternary-complex mechanism, with initial binding of NADPH before glucose and final release of NADP+.
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Project description:The effects of modifiers (NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol and p-nitrobenzaldehyde) on the hydrolysis of p-nitrophenyl (PNP) pivalate (PNP trimethylacetate) catalysed by cytoplasmic aldehyde dehydrogenase are reported. In each case a different inhibition pattern is obtained to that observed when the substrate is PNP acetate; for example, propionaldehyde and chloral hydrate competitively inhibit the hydrolysis of PNP acetate, but are mixed inhibitors with PNP pivalate. The kinetic results can be rationalized in terms of different rate-determining steps: acylation of the enzyme in the case of the pivalate but acyl-enzyme hydrolysis for the acetate. This is confirmed by stopped-flow studies, in which a burst of p-nitrophenoxide is observed when the substrate is PNP acetate, but not when it is the pivalate. PNP pivalate inhibits the dehydrogenase activity of the enzyme competitively with the aldehyde substrate; this is most simply explained if the esterase and dehydrogenase reactions occur at a common enzymic site.
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Project description:We conducted RNA sequcing of the homozygous and heterozygous of ALDH2*2 by using human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs)
2023-03-30 | GSE184419 | GEO
Project description:Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs.
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Project description:An improved procedure for purifying aldehyde reductase is described. Utilization of Blue Dextran--Sepharose 4B and elimination of hydroxyapatite chromatography greatly improves the yield and ease of purification. Starting with 340 g of kidney tissue (two pig kidneys) approx. 50 mg of purified reductase may be routinely and reproducibly obtained. The purified reductase was used to establish the kinetic reaction mechanism of the enzyme. Initial-velocity analysis and product-inhibition data revealed that pig kidney aldehyde reductase follows an Ordered Bi Bi reaction mechanism in which NADPH binds first before D-glyceraldehyde. The limiting Michaelis constants for D-glyceraldehyde and NADPH were 4.8 +/- 0.7 mM and 9.1 +/- 2.1 micrometer respectively. The mechanism is similar to that of another monomeric oxidoreductase, octopine dehydrogenase, towards which aldehyde reductase exhibits several similarities, but differs from that of other aldehyde reductases. Phenobarbital is a potent inhibitor of aldehyde reductase, inhibiting both substrate and cofactor non-competitively (Ki = 80.4 +/- 10.5 micrometer and 66.9 +/- 1.6 micrometer respectively). Barbiturate inhibition seems to be a common property of NADPH-dependent aldehyde reductases.
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