Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents.

Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2'-dithiodipyridine and 4,4'-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4'-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2'-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5'-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.     

Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase.

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.     

Further studies of the action of disulfiram and 2,2''-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase.

1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2'-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2'-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.     

Effects of diethylstilbestrol, 2,2'-dithiodipyridine, and chloral hydrate on the esterase activity of sheep liver cytoplasmic aldehyde dehydrogenase

The binding of diethylstilbestrol (DES) to aldehyde dehydrogenase (ALDH) has a very similar effect on the dehydrogenase activity of the enzyme as has modification of the enzyme by 2,2'-dithiodipyridine [Kitson, T.M. (1982) Biochem. J. 207, 81-89]. The latter modification may occur at the site of the esterase activity of the enzyme [Kitson, T.M. (1985) Biochem. J. 228, 765-767]. This suggests that DES might be a competitive inhibitor of the esterase reaction. However, in the absence of oxidized nicotinamide adenine dinucleotide (NAD+) or reduced nicotinamide adenine dinucleotide (NADH), and at low concentrations of substrate (4-nitrophenyl acetate, PNPA), DES is a potent partial noncompetitive inhibitor. It is concluded therefore that DES binds at a site different from the esterase active site and that the enzyme-DES complex retains some ability to act as an esterase. High concentrations of PNPA appear to displace DES from its binding site. In the presence of NAD+, DES is a weaker inhibitor, and in the presence of NADH, DES has very little effect. Esterase activity is enhanced by NADH when PNPA concentrations are high but is inhibited when they are low. The rate of reaction of ALDH with 2,2'-dithiodipyridine is only slightly reduced by DES, suggesting that the site at which thiol modifiers react and the DES binding site are different. When ALDH is modified by 2,2'-dithiodipyridine, it has reduced esterase activity, which declines further as the modified enzyme loses its 2-thiopyridyl label. In the presence of NAD+, chloral hydrate is a simple competitive inhibitor of the esterase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of aldehyde dehydrogenase with dicyclohexylcarbodiimide: Separation of dehydrogenase from esterase activity

Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide (DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD⁺ abolished the burst. TheK _m for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity was the result of covalent bond formation. Protection by NAD⁺ and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E1 isozyme. The results suggested that some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC.     

Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase.

The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2'-Dithiodipyridine is an activator of the enzyme.     

The action of cytoplasmic aldehyde dehydrogenase on methyl p-nitrophenyl carbonate and p-nitrophenyl dimethylcarbamate.

Cytoplasmic aldehyde dehydrogenase catalyses the hydrolysis of methyl p-nitrophenyl (PNP) carbonate at an appreciable rate that is markedly stimualted by NAD+ or NADH. The nuleotides accelerate the rate-limiting hydrolysis of the acyl-enzyme intermediate while slowing the observed burst of p-nitrophenoxide production. With PNP dimethylcarbamate the enzyme catalyses the slow release of approx. 1 molecule of p-nitrophenoxide per tetrameric enzyme molecule; during the reaction the enzyme becomes effectively inactivated, as the rate of hydrolysis of the acyl-enzyme is virtually zero. The presence of NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol or disulfiram slows the reaction of enzyme with PNP dimethylcarbamate. The reaction appears to be dependent on a group of pKa 7.6, possibly a cysteine residue. The effect of PNP dimethylcarbamate on the dehydrogenase activity of the enzyme is consistent with there being a single type of active site for the enzyme's dehydrogenase and esterase activities. Steric and electronic factors that govern reaction of the enzyme with PNP substrates are discussed.     

The action of progesterone and diethylstilboestrol on the dehydrogenase and esterase activities of a purified aldehyde dehydrogenase from rabbit liver.

A steroid-sensitive aldehyde dehydrogenase (EC 1.2.1.3) was purified from rabbit liver and is homogeneous by the criterion of electrophoresis in polyacrylamide gels with or without sodium dodecyl sulphate. The enzyme is tetrameric, of subunit mo.wt. 48 300, and contains no tightly bound zinc. The fluorescence of the protein is decreased in the presence of progesterone, which is inhibitory to the reactions catalysed by the enzyme. When NADH is bound to the enzyme, the fluorescence of the coenzyme is augmented to an extent independent of the presence of steroids or acetaldehyde. The purified enzyme catalyses the oxidation of acetaldehyde and glucuronolactone, and the hydrolysis of 4-nitrophenyl acetate. Each of these reactions is inhibited by progesterone in such a manner as to suggest the formation of a catalytically active enzyme-hormone complex. Diethylstilboestrol inhibits the hydrolysis of esters by this enzyme, but stimulates the oxidation of aldehydes, except at low aldehyde concentrations; the ligand is then inhibitory. NADH inhibits the hydrolysis of 4-nitrophenyl acetate by the enzyme in a partially competitive fashion.     

Studies on the oligomeric structure of yeast aldehyde dehydrogenase by cross-linking with bifunctional reagents.

The molecular w:ight of yeast aldehyde dehydrogenase determined by sucrose density gradient centrifugation was 207,000 +/- 13,000. The enzyme activity was proportional to the enzyme concentration in the range of 2 X 10(-11) M to 1 X 10(-7) M. Cross-linking patterns obtained with yeast aldehyde dehydrogenase after treatment with a series of diimidoesters of increasing chain lengths with different reaction times resulted in the appearance of tetramers as the largest cross-linked product of the enzyme subunits. The molecular weights of its monomer, dimer, trimer, and tetramer were, 57,000, 114,000, 171,000, and 228,000, respectively, as estimated from their mobilities on SDS-electrophoresis. In tetramers monomers are probably assembled in a heterologous square arrangement.     

Multifunctionality of liver alcohol dehydrogenase. Studies of aldehyde dehydrogenase activity

Kinetic studies of the liver alcohol dehydrogenase catalyzed dehydrogenation of aldehydes were carried out over a wide range of octanal concentrations. The effect of specific inhibitors of liver alcohol dehydrogenase on aldehyde dehydrogenase activity was examined. The results were consistent with a steady-state random mechanism with the formation of the ternary E · NADH octanal complex at low temperatures. This ternary complex becomes inconspicuous at high temperatures. The aldehyde dehydrogenase activity was found to associate with all ethanol-active isozymes. The dual dehydrogenase reactions are catalyzed by the same molecule, presumably in the region of the same domain. However, the two activities respond differently to structural changes.     

Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD(+) and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD(+) and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD(+), estimates of 2x10(5) litre.mol(-1).s(-1) and 2s(-1) were obtained for k(+1) and k(-1) respectively. Although k(+3) could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s(-1), k(+2) and k(-2) could not be determined uniquely, but could be related by the equation: (k(-2)+k(+3))/k(+2) =50x10(-6)mol.litre(-1). No significant isotope effect was observed when [1-(2)H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k(-2)+k(+3))/k(+2)=2.3x10 (-3)mol.litre(-1) and k(+3) is 23s(-1). When [1,2,2,2-(2)H]acetaldehyde was used, no isotope effect was observed on k(+3), but there was a significant effect on k(+2) and k(-2). A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.     

The harmful action of ethanol on the liver: the role of alcohol dehydrogenase and aldehyde dehydrogenase

White rats were divided into water-preferring (WP) and ethanol-preferring (EP) groups, on the basis of their preferable drink: either water or 15% solution of ethanol. Each of these groups was then subdivided into groups which were given to drink for 1 year 15% solution of ethanol (ethanol-treated) or water (controls). Alcohol dehydrogenase/aldehyde dehydrogenase activity ratios (ADH/AlDH) in livers of WP controls were considerably higher than those in EP controls. The difference in ADH/AlDH has somewhat decreased after ethanol treatment. However, this ratio remained the highest in the WP alcohol-treated group. The signs of proteinic and lipid dystrophy of the liver in alcohol-treated WP rats were expressed much more clearly than in all other groups. It is concluded that in the liver of animals with a high ADH/AlDH ratio there are favourable conditions for accumulation of a toxic hepatocyte-damaging acetaldehyde.     

Pesticide induced up-regulation of esterase and aldehyde dehydrogenase in indigenous Bacillus spp.

During bacterial growth an increased mRNA level is usually linked with higher rate of metabolism related to biodegradation of an unusual compound. In this study, quantitative gene expression data derived from mRNA level reveal the presence of pesticide degrading genes in 6 bacterial isolates showing biodegradation of cypermethrin (SG2, SG4), sulfosulfuron (SA2, Sulfo3), and fipronil (FA3, FA4). A correlation existed between the level of esterase coding mRNA and mineralization of cypermethrin in SG2 and SG4. Similarly the level of EST coding mRNA increased with biodegradation of fipronil and sulfosulfuron in FA3, FA4, SA2, and Sulfo3. Expression of est gene was observed in all the bacterial strains, but their level of expression was different. Bacterial strains SG4 and Sulfo3 showed higher level of est gene expression as compared to SG2, FA3, FA4, and SA2 and was in the range of approximately 30- to 60-fold, respectively, in comparison to control. Expression of genes for aldehyde dehydrogenase was observed in SG4 and Sulfo3. We report co expression of aldh (1000?bp) and est (～550?bp) genes at the same time of pesticide induction/biodegradation.     

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase.

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.     

Kinetics of p-nitrophenyl pivalate hydrolysis catalysed by cytoplasmic aldehyde dehydrogenase.

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.     

Effect of SH-reagents on aldehyde dehydrogenase activity of the rat liver

SH-reagents: tetraethylthiuram disulphide (TETD), 5,5'-dithiobisnitrobenzoic acid (DTNB), p-chloromercurybenzoate (p-ChMB), N-ethylmaleimide (NEM) were studied for their effect on the aldehyde dehydrogenase activity of mitochondrion (isoenzymes I and II) and microsome (isoenzyme II) fractions of the rat liver. TETD is established to inhibit isoenzyme I and isoenzyme II activity of mitochondrial aldehyde dehydrogenase by 100 and 50%, respectively, and the microsomal enzyme activity by 20%. DTNB and NEM inhibit 30-50% of the activity in two isoforms of mitochondrial aldehyde dehydrogenase having no effect on the enzymic activity in microsomes; p-ChMB inhibits completely the activity of the enzyme under study both in the mitochondrial and microsomal fractions. A conclusion is drawn that SH-groups are very essential for manifestation of the catalytic activity in the NAD+-dependent aldehyde dehydrogenase from mitochondrial and microsomal fractions.