Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases.

BACKGROUND: . Enzymes of the aldehyde dehydrogenase family are required for the clearance of potentially toxic aldehydes, and are essential for the production of key metabolic regulators. The cytosolic, or class 1, aldehyde dehydrogenase (ALDH1) of higher vertebrates has an enhanced specificity for all-trans retinal, oxidising it to the powerful differentiation factor all-trans retinoic acid. Thus, ALDH1 is very likely to have a key role in vertebrate development. RESULTS: . The three-dimensional structure of sheep ALDH1 has been determined by X-ray crystallography to 2.35 A resolution. The overall tertiary and quaternary structures are very similar to those of bovine mitochondrial ALDH (ALDH2), but there are important differences in the entrance tunnel for the substrate. In the ALDH1 structure, the sidechain of the general base Glu268 is disordered and the NAD+ cofactor binds in two distinct modes. CONCLUSIONS: . The submicromolar Km of ALDH1 for all-trans retinal, and its 600-fold enhanced affinity for retinal compared to acetaldehyde, are explained by the size and shape of the substrate entrance tunnel in ALDH1. All-trans retinal fits into the active-site pocket of ALDH1, but not into the pocket of ALDH2. Two helices and one surface loop that line the tunnel are likely to have a key role in defining substrate specificity in the wider ALDH family. The relative sizes of the tunnels also suggest why the bulky alcohol aversive drug disulfiram reacts more rapidly with ALDH1 than ALDH2. The disorder of Glu268 and the observation that NAD+ binds in two distinct modes indicate that flexibility is a key facet of the enzyme reaction mechanism.

Studies on the chymotrypsin-catalysed hydrolysis of resorufin acetate and resorufin bromoacetate.

Resorufin bromoacetate is a substrate that is rapidly hydrolysed by chymotrypsin. The reaction shows a pre-steady-state burst phase that may be observed by stopped flow spectrophotometry if precautions are taken against spontaneous hydrolysis of the substrate. The strongly activating effect that the presence of the bromine atom has on the adjacent carbonyl group is reflected in the relative sizes of the kcat values for resorufin bromoacetate and resorufin acetate (e.g., 740 to 1, at pH 6) and the burst rate constants (e.g., 350 to 1, at pH 7 using 0.1 mM substrate). The pH-dependence of kcat for both substrates shows the involvement of an enzymic group of pKa about 7. With resorufin bromoacetate, a burst and a steady-state rate are still observable at pH 3.0. Unlike the case with aldehyde dehydrogenase, resorufin bromoacetate does not act as an inactivator of chymotrypsin and there is little or no incorporation of covalently-linked label when chymotrypsin and resorufin bromoacetate are incubated together. The different modes of behaviour of the two enzymes are attributable to the 'hard' or 'soft' character of the attacking enzymic nucleophilic groups.

Studies of the action of symmetrical and mixed disulphide thiol-modifying reagents on the esterase activity of cytoplasmic aldehyde dehydrogenase.

The effect of various thiol-modifying reagents on the esterase activity of sheep liver cytoplasmic aldehyde dehydrogenase is reported here. Both symmetrical reagents (disulfiram, 2,2'- and 4,4'-dithiodipyridines) and unsymmetrical reagents (methyl diethylthiocarbamyl disulphide, methyl 2- and 4-pyridyl disulphides) were investigated. The results suggest that all the modifiers react to varying extents with a pair of enzymic thiol groups ('A' and 'B'), and that the more specifically group 'A' is modified, the more the enzyme is inactivated. This supports the idea that group 'A' may be the essential nucleophile in the reaction catalysed by aldehyde dehydrogenase. Modification of group 'B' may or may not reduce the esterase activity depending on the nature of the label introduced. The results of the present experiments and of previous similar experiments concerning the dehydrogenase activity of the enzyme are consistent with the proposal that a common active site is responsible for both esterase and dehydrogenase activities.

The effect of quercetin, a widely distributed flavonoid in food and drink, on cytosolic aldehyde dehydrogenase: a comparison with the effect of diethylstilboestrol.

Quercetin is a flavonoid found in red wine and many other dietary sources. Observations concerning the state of ionisation and the stability of the compound over a range of pH are presented. Quercetin is a potent inhibitor of cytosolic aldehyde dehydrogenase at physiological pH when the concentration of either the substrate or the cofactor is relatively low, but it has an activatory effect when the concentrations of substrate and cofactor are both high (1 mM). Gel filtration experiments show that quercetin binds very tightly to the enzyme under conditions where the compound is neutral and when it is ionised. The binding is less in the presence of NAD(+). Quercetin cuts down the ability of the resorufin anion to bind to the enzyme. The observations are explained by a model in which quercetin binds competitively to both the coenzyme-binding site and the aldehyde-binding site; binding in the latter location, when the enzyme is in the form of the E-NADH complex, accounts for the activation. The effects of quercetin are significantly different in some respects from those of diethylstilboestrol; this is explained by the latter being able to bind to the aldehyde site but not the NAD(+) site. The possibility that quercetin may affect aldehyde dehydrogenase in vivo is discussed.

Crystallization and preliminary X-ray diffraction studies on cytosolic (class 1) aldehyde dehydrogenase from sheep liver.

The cytosolic (Class 1) aldehyde dehydrogenase (AlDH) from sheep liver has been crystallized in a form suitable for X-ray diffraction studies. The crystals, grown by vapour diffusion using 6.5 to 7.5% methoxypolyethylene glycol 5000 as precipitant, at pH 6.5, are orthorhombic with cell dimensions a = 80.7, b = 92.5, c = 151.6 A, space-group P2(1)2(1)2(1), and one dimer in the asymmetric unit. The crystals diffract to at least 2.8 A resolution. Although unmodified AlDH crystallized readily, a key factor in obtaining diffraction-quality crystals was the covalent attachment of an active site reporter group, provided by 3,4-dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one.

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin, resveratrol and diethylstilbestrol.

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD(+)-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD(+) concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD(+), by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.

The effect of cephalosporin antibiotics on alcohol metabolism: a review.

A review is made of the pharmacological, biochemical and chemical aspects of the unpleasant 'Antabuse-like' reaction that may be induced in drinkers of alcohol by pre-treatment with certain beta-lactam antibiotics with a 1-methyltetrazole-5-thiol sidechain (such as moxalactam, cefamandole and cefoperazone). The symptoms are due to abnormally elevated blood acetaldehyde levels consequent upon the inactivation of hepatic aldehyde dehydrogenase. There is very little direct effect of the antibiotics on this enzyme and therefore it is concluded that a reactive metabolite of the antibiotics' essential sidechain is responsible for the reaction. A likely candidate for this active species is either the symmetrical disulphide 5,5'-dithiobis(1-methyltetrazole) formed by oxidation of 1-methyltetrazole-5-thiol, or the related mixed disulphide, methyl 5-(1-methyltetrazolyl) disulphide. The first of these is a potent inactivator of cytoplasmic aldehyde dehydrogenase only, the second affects both cytoplasmic and mitochondrial isoenzymes. 1-Methyltetrazole-5-thiol or derivatives have the potential to be used therapeutically as 'anti-alcohol' compounds in the same way as disulfiram (Antabuse) or calcium cyanamide.

Modification of thiol groups in cytoplasmic aldehyde dehydrogenase.

It is proposed that cytoplasmic aldehyde dehydrogenase possesses a pair of important reactive thiol groups, A and B. Group A is labelled by disulfiram and the enzyme is inactivated; subsequently group B displaces the dithiocarbamate label and an enzymic disulphide is formed. On the other hand, it appears that group B is labelled by 2,2'-dithiodipyridine resulting in activation of the enzyme. Again, the label (2-thiopyridone) is later displaced, this time presumably by group A, giving rise to loss of enzymic activity and formation of the same disulphide species as is produced by disulfiram. Methyl diethylthiocarbamyl disulphide and methyl 2-pyridyl disulphide supply the same label (MeS-) but the first compound inactivates the enzyme while the second activates it. It is concluded that the first of these reagents modifies group A and the second group B. It appears that methyl 4-pyridyl disulphide may react non-specifically with both groups A and B. Group A is a possible candidate for a catalytically essential nucleophile in the actions of aldehyde dehydrogenase.

Reinvestigation of the chemical reaction between disulfiram and ethanol.

A claim that disulfiram and ethanol react to produce a quaternary ammonium compound, and that this product may be involved in the pharmacogenesis of the disulfiram-ethanol reaction, is shown to be in error.

On the probability of implanted disulfiram's causing a reaction to ethanol.

Calculations indicate that patients with implanted disulfiram are unlikely to experience a pharmacologically induced disulfiram--ethanol reaction.

Drugs which interfere with alcohol metabolism.

A review is made of compounds which produce antagonism to alcohol. Some of these drugs are used purposefully in alcoholism therapy to curtail drinking; others, which are administered for quite unrelated reasons in the treatment of different medical conditions, may also cause unpleasant symptoms if alcohol is subsequently ingested.