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

ABSTRACT: 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.

SUBMITTER: Kitson TM

PROVIDER: S-EPMC1135617 | biostudies-other | 1989 Jan

REPOSITORIES: biostudies-other

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

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.

| S-EPMC1135616 | biostudies-other

The binding of NADH to cytoplasmic aldehyde dehydrogenase after modification with p-nitrophenyl dimethylcarbamate.

Project description:Cytoplasmic aldehyde dehydrogenase may be modified by reaction with p-nitrophenyl dimethylcarbamate to give a stable E-X-CO-NMe2 species that is an analogue of the usual labile acyl-enzyme involved in the enzyme's reactions. [X is derived from an enzymic nucleophilic group.] This species still contains the tightly bound NADH that is present in the native enzyme. When further NADH binds to E-X-CO-NMe2 its fluorescence is enhanced over 4 times more than when it binds to unmodified enzyme; this fluorescence is completely unaffected by high propionaldehyde concentration and only slightly affected by p-nitrobenzaldehyde. The modified species has 1.0 NADH binding site in the absence of Mg2+ and 1.67 sites in its presence. The rate of dissociation of E-X-CO-NMe2.NADH is biphasic (k 3.4 and 1.8 min-1) and is considerably lower than that of E.NADH; the presence of Mg2+ slows the process even more (k 0.47 and 0.37 min-1). The implications of these studies towards a greater understanding of the nature of aldehyde dehydrogenase-catalysed reactions are discussed.

| S-EPMC1135618 | biostudies-other

Aldehyde dehydrogenase homologous folate enzymes: Evolutionary switch between cytoplasmic and mitochondrial localization.

Project description:Cytosolic and mitochondrial 10-formyltetrahydrofolate dehydrogenases are products of separate genes in vertebrates but only one such gene is present in invertebrates. There is a significant degree of sequence similarity between the two enzymes due to an apparent origin of the gene for the mitochondrial enzyme (ALDH1L2) from the duplication of the gene for the cytosolic enzyme (ALDH1L1). The primordial ALDH1L gene originated from a natural fusion of three unrelated genes, one of which was an aldehyde dehydrogenase. Such structural organization defined the catalytic mechanism of these enzymes, which is similar to that of aldehyde dehydrogenases. Here we report the analysis of ALDH1L1 and ALDH1L2 genes from different species and their phylogeny and evolution. We also performed sequence and structure comparison of ALDH1L enzymes possessing aldehyde dehydrogenase catalysis to those lacking this feature in an attempt to explain mechanistic differences between cytoplasmic ALDH1L1 and mitochondrial ALDH1L2 enzymes and to better understand their functional roles.

| S-EPMC4414694 | biostudies-literature

Aldehyde dehydrogenase catalyses acetaldehyde formation from 4-nitrophenyl acetate and NADH.

Project description:Incubation of sheep liver cytoplasmic aldehyde dehydrogenase with the substrate 4-nitrophenyl [14C]acetate in the presence of NADH leads to the formation of 14C-labelled acetaldehyde. This observation strongly supports the idea that the esterase and dehydrogenase activities of the enzyme occur at the same site and involve the intermediacy of a common acyl-enzyme.

| S-EPMC1147177 | biostudies-other

Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol.

Project description:BackgroundIn a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol.MethodsTriple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol.ResultsIn the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol.ConclusionThis study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol.General significanceThe present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.

| S-EPMC3669398 | biostudies-literature

A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase.

Project description:1. p-Nitrophenyl (PNP) acetate and propionate show a burst of p-nitrophenoxide release when their hydrolysis is catalysed by sheep liver cytosolic aldehyde dehydrogenase. This is not seen in the presence of NAD+ or NADH, implying a change in ratedetermining step. 2. 6-Nitrodihydrocoumarin (6-NDC) shows no burst of absorbance in the visible region. We propose that the pKa of the transient "reporter group' produced during the hydrolysis of this lactone is high (approx. 10) and that the incipient covalently linked p-nitrophenoxide moiety is protonated immediately on formation. The small burst seen in the hydrolysis of 5-nitro-2-coumaranone (5-NC) suggests that the pKa of its reporter group is about 8.5. 3. NADH markedly enhances the steady-state rate with the lactones. 5-NC shows a large rapid burst of colour development in the presence of NADH; this implies that NADH decreases the pKa of the reporter group to 7-7.5. 4. In the presence of NAD+, 5-NC and 6-NDC give an unusual "negative burst' in the stopped-flow traces. We propose that, under these circumstances, acylation of the enzyme is extremely fast and that the first event seen in the stopped-flow traces is protonation of the reporter group. NAD+ also greatly increases the steady-state rate. 5. With the lactones in the presence of NADH, the kcat value (nearly 6 s-1), a measure of the deacylation rate, is compatible with the single-site model for dehydrogenase and esterase activities.

| S-EPMC1217326 | biostudies-other

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

Project description: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.

| S-EPMC1158291 | biostudies-other

Evidence that the cytoplasmic aldehyde dehydrogenase-catalysed oxidation of aldehydes involves a different active-site group from that which catalyses the hydrolysis of 4-nitrophenyl acetate.

Project description:Acylation of the aldehyde dehydrogenase.NADH complex by acetic anhydride leads to the production of acetaldehyde and NAD+. By monitoring changes in nucleotide fluorescence, the rate constant for acylation of the active site of the *enzyme.NADH complex was found to be 11 +/- 3 s-1. The rate of acylation by acetic anhydride at the group that binds aldehydes on the oxidative pathway is clearly rapid enough to maintain significant steady-state concentrations of the required active-site-acylated *enzyme.NADH intermediate despite the rapid hydrolysis of this *enzyme.acyl.NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman & Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase.NADH complex by 4-nitrophenyl acetate also occurs rapidly with a rate constant of 10.9 +/- 0.6 s-1, even under the most extreme trapping conditions only very small amounts of acetaldehyde are detected [Loomes & Kitson (1986) Biochem. J. 235, 617-619]. Furthermore enzyme-catalysed hydrolysis of 4-nitrophenyl acetate is limited by the rate of deacylation of a group on the enzyme (0.4 s-1), which is an order of magnitude less than deacylation of the group at the active site (5-10 s-1). It is concluded that the enzyme-catalysed 4-nitrophenyl ester hydrolysis involves a group on the enzyme that is different from the active-site group that binds aldehydes on the normal oxidative pathway.

| S-EPMC1135169 | biostudies-other

Mechanism of inactivation of sheep liver cytoplasmic aldehyde dehydrogenase by disulfiram.

Project description:Stoicheiometric amounts of [14C]disulfiram react rapidly with sheep liver cytoplasmic aldehyde dehydrogenase to give loss of catalytic activity and incorporation of the expected amount of radioactivity. In a subsequent slower reaction the label is lost from the enzyme without re-emergence of enzymic activity. The results imply that in vivo disulfiram may act as an oxidation-reduction catalyst for the inactivation of aldehyde dehydrogenase.

| S-EPMC1152162 | biostudies-other

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

Project description: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.

| S-EPMC1183679 | biostudies-other

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