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Probing the active site of cytoplasmic aldehyde dehydrogenase with a chromophoric reporter group.

ABSTRACT: 3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one ('DMNB') reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pKa of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD+ to the modified enzyme, the pKa falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD+. The modified enzyme binds NAD+ very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD+ when NADH is added.

SUBMITTER: Kitson TM

PROVIDER: S-EPMC1138117 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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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

High concentrations of aldehydes slow the reaction of cytoplasmic aldehyde dehydrogenase with thiol-group modifiers.

Project description:High concentrations of aldehydes slow the inactivation of cytoplasmic aldehyde dehydrogenase by disulfiram and also slow the reaction of the enzyme with 2,2'-dithiodipyridine. It is concluded that a low-affinity aldehyde-binding site is probably the site at which thiol-group modifiers react with aldehyde dehydrogenase, as well as being the active site for hydrolysis of 4-nitrophenyl acetate.

| S-EPMC1145050 | 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

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:In 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 ?ALDH2+?ALDH5 or ?ALDH1+?ALDH5 could grow on ethanol. However, the double deleted strain ?ALDH1+?ALDH2 did not grow in ethanol.Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol.In 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 (?ALDH1+?ALDH2+?ALDH5) 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.This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol.The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.

| S-EPMC3669398 | biostudies-literature

NAD+ promotes assembly of the active tetramer of aldehyde dehydrogenase 7A1.

Project description:Nicotinamide adenine dinucleotide (NAD) is the redox cofactor of many enzymes, including the vast aldehyde dehydrogenase (ALDH) superfamily. Although the function of NAD(H) in hydride transfer is established, its influence on protein structure is less understood. Herein, we show that NAD+ -binding promotes assembly of the ALDH7A1 tetramer. Multiangle light scattering, small-angle X-ray scattering, and sedimentation velocity all show a pronounced shift of the dimer-tetramer equilibrium toward the tetramer when NAD+ is present. Furthermore, electron microscopy shows that cofactor binding enhances tetramer formation even at the low enzyme concentration used in activity assays, suggesting the tetramer is the active species. Altogether, our results suggest that the catalytically active oligomer of ALDH7A1 is assembled on demand in response to cofactor availability.

| S-EPMC6188814 | biostudies-literature

A study of the role of the reactive thiol group of rabbit muscle creatine kinase with a chromophoric reporter group.

Project description:Substrate- and ligand-induced conformational changes were studied in a series of thiol-modified derivatives of rabbit muscle creatine kinase that retained different amounts of enzymic activity. The results indicate that the 'reactive' thiol group of the enzyme is required for the conformational changes associated with formation of a 'transition-state analogue' complex.

| S-EPMC1184157 | biostudies-other

Aldehyde dehydrogenase. An enzyme with two distinct catalytic activities at a single type of active site.

Project description:The evidence for and against the esterase and dehydrogenase active sites of aldehyde dehydrogenase being topologically distinct is examined. It is found that all the evidence (including all that previously amassed by others in favour of distinct binding domains) is actually consistent with, and in favour of, a single type of catalytic site having both activities. The existence of separate high-Km modulating sites for the enzyme is also questioned.

| S-EPMC1152610 | biostudies-other

Structural and biochemical consequences of pyridoxine-dependent epilepsy mutations that target the aldehyde binding site of aldehyde dehydrogenase ALDH7A1.

Project description:In humans, certain mutations in the gene encoding aldehyde dehydrogenase 7A1 are associated with pyridoxine-dependent epilepsy (PDE). Understanding the impact of PDE-causing mutations on the structure and activity of ALDH7A1 could allow for the prediction of symptom-severity and aid the development of patient-specific medical treatments. Herein, we investigate the biochemical and structural consequences of PDE missense mutations targeting residues in the aldehyde substrate binding site: N167S, P169S, A171V, G174V, and W175G. All but G174V could be purified for biochemical and X-ray crystallographic analysis. W175G has a relatively mild kinetic defect, exhibiting a fivefold decrease in kcat with no change in Km . P169S and N167S have moderate defects, characterized by catalytic efficiencies of 20- and 100-times lower than wild-type, respectively. A171V has a profound functional defect, with catalytic efficiency 2000-times lower than wild-type. The crystal structures of the variants are the first for any PDE-associated mutant of ALDH7A1. The structures show that missense mutations that decrease the steric bulk of the side chain tend to create a cavity in the active site. The protein responds by relaxing into the vacant space, and this structural perturbation appears to cause misalignment of the aldehyde substrate in W175G and N167S. The P169S structure is nearly identical to that of the wild-type enzyme; however, analysis of B-factors suggests the catalytic defect may result from altered protein dynamics. The A171V structure suggests that the potential for steric clash with Val171 prevents Glu121 from ion pairing with the amino group of the aldehyde substrate. ENZYMES: Aldehyde dehydrogenase 7A1 (EC1.2.1.31). DATABASES: Coordinates have been deposited in the Protein Data Bank under the following accession codes: 6O4B, 6O4C, 6O4D, 6O4E, 6O4F, 6O4G, 6O4H.

| S-EPMC6944758 | biostudies-literature

The Mo-Se active site of nicotinate dehydrogenase.

Project description:Nicotinate dehydrogenase (NDH) from Eubacterium barkeri is a molybdoenzyme catalyzing the hydroxylation of nicotinate to 6-hydroxynicotinate. Reactivity of NDH critically depends on the presence of labile (nonselenocysteine) selenium with an as-yet-unidentified form and function. We have determined the crystal structure of NDH and analyzed its active site by multiple wavelengths anomalous dispersion methods. We show that selenium is bound as a terminal Mo=Se ligand to molybdenum and that it occupies the position of the terminal sulfido ligand in other molybdenum hydroxylases. The role of selenium in catalysis has been assessed by model calculations, which indicate an acceleration of the critical hydride transfer from the substrate to the selenido ligand in the course of substrate hydroxylation when compared with an active site containing a sulfido ligand. The MoO(OH)Se active site of NDH shows a novel type of utilization and reactivity of selenium in nature.

| S-EPMC2708692 | biostudies-literature

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

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