Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.1 (alcohol dehydrogenase)
9,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Toluene and its metabolites have been studied with respect to their reactive oxygen species-enhancing potential in isolated systems and in vivo. The induction of reactive oxygen species (ROS) production was assayed using the probe 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). Intraperitoneal injection of toluene, benzyl alcohol or benzaldehyde caused a significant elevation in the rate of ROS formation within hepatic mitochondrial fractions (P2). In the brain, only toluene induced ROS formation, while benzyl alcohol and benzaldehyde did not have any effect. Glutathione (GSH) levels were depressed in liver and brain regions from toluene-treated rats. However, no such depression was evident in brains treated with toluene metabolites. P2 fractions from phenobarbital-pretreated rats exhibited a heightened ROS response when challenged with toluene, in vitro. Pretreatment of rats in vivo with 4-methylpyrazole, an alcohol dehydrogenase inhibitor, or sodium cyanamide, an aldehyde dehydrogenase inhibitor, prior to exposure to toluene, caused a significant decrease and increase, respectively, in toluene-stimulated rates of ROS generation in the CNS and liver. Electron spin resonance spectroscopy, employing the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was conducted. Incubation of the spin trap with P2 fractions and toluene or benzaldehyde elicited a spectrum corresponding to the hydroxyl radical. Incubation of benzaldehyde with aldehyde dehydrogenase produced a strong signal that was blocked completely by superoxide dismutase and inhibited partially by catalase, suggesting the presence of superoxide radicals and the involvement of the iron-catalyzed Haber-Weiss reaction leading to the production of hydroxyl radicals. Thus, ROS generation during toluene catabolism may occur at two steps: cytochrome P450 oxidation and aldehyde dehydrogenase oxidation. In addition, GSH may play an important role in protection against the induction of ROS generation in the CNS and liver following exposure to toluene.
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PMID:Free radical induction in the brain and liver by products of toluene catabolism. 839 73

Primary and secondary kD/kT and kH/kT kinetic isotope effects have been studied as a probe of hydrogen tunneling in the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase (LADH). In the case of the wild-type enzyme, isotope effects at 25 degrees C do not clearly support hydrogen tunneling; this result is consistent with a reaction rate that is partially limited by the release of product benzaldehyde. The three-dimensional structure for LADH was used to design site-directed mutations in an effort to enhance the rate of the product release step and to "unmask" tunneling. Substitutions that increased the size of the alcohol binding pocket resulted in minor changes in isotope effects. By contrast, reduction in the size of the alcohol binding pocket through substitution at residues 57 and 93, which are in van der Waals contact with bound alcohol substrate, produced a clear demonstration of protium tunneling from the breakdown of the semiclassical relationship between kD/kT and kH/kT isotope effects. The temperature dependence of kD/kT isotope effects has also been pursued, leading to the conclusion that tunneling does, in fact, occur in the reaction catalyzed by wild-type LADH. Despite the unmasking of protium tunneling in site-directed mutants, substitutions that decrease the size of the alcohol pocket appear to result in less extensive tunneling in the hydride transfer. It is noteworthy that the mutant enzyme (Leu57-->Phe), which shows the greatest evidence of tunneling, has the same catalytic efficiency (Vmax/Km) as the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Unmasking of hydrogen tunneling in the horse liver alcohol dehydrogenase reaction by site-directed mutagenesis. 850 71

The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed by horse liver alcohol dehydrogenase (HLADH) has been investigated using the HLADH structure at 2.1 A resolution with NAD+ and pentafluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations with HLADH were obtained by a best-fit superimposition of benzaldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II)ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(II) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initio-derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21,40-56]. Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MD1) enzyme with NAD+, benzaldehyde, and zinc-ligated HO-in the active site; (MD2) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxygen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempirical PM3 calculations. Using PM3, model systems were calculated of ground states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc-bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resided at a distance of 2.8-5.5 A from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 transition state for attack of the Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, which has an O...C distance of 1.877 A. HLADH catalysis of the aldehyde hydration would require very little motion aside from that in the ground state. Two simulations of benzaldehyde hydrate ligated to zinc (MD2 and MD3) both showed close approach of the aldehyde hydrate hydrogen to NAD+C4, varying from 2.3 to 3.3 A, seemingly favorable for the hydride transfer reaction. The MD2 configuration does not allow proton shuttling. On the other hand, when the pro-S oxygen is ligated to zinc (MD3), the proton on the pro-R oxygen averages 2.09 A from the hydroxyl oxygen of Ser48 such that initiation of shuttling of protons via Ser48 to the ribose 2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen is sterically favorable. PM3 calculations suggest that this proton shuttle represents a stepwise reaction which occurs subsequent to hydride transfer. The PM3 transition state for hydride transfer based on the MD3 configuration has the transferring hydride 1.476 A from C4 of NAD+ and 1.433 A from the aldehyde alpha-carbon.
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PMID:Mechanism of aldehyde oxidation catalyzed by horse liver alcohol dehydrogenase. 870 51

Two highly fluorogenic aldehydes, 7-methoxy-1-naphthaldehyde (MONAL-71) and 6-methoxy-2-naphthaldehyde (MONAL-62), were examined as indicators of the aldehyde dehydrogenase (ALDH) activity in human tissue homogenates and accessible body fluids. Both compounds were previously found to be excellent substrates for the ALDH from erythrocytes and for the purified class I (cytosolic) ALDH from human liver. By contrast, only MONAL-62, but not the isomeric MONAL-71, was oxidized by class III ALDH present in human saliva. The apparent Km for the former compound reacting with salvia ALDH is 0.24 microM, with the reaction rate (Vmax) close to that of benzaldehyde oxidation. There is also a fully competitive inhibition of the fluorogenic oxidation of the MONAL-62 by benzaldehyde. Both NAD+ and NADP+ can be used as oxidants in this reaction, with comparable rates, a fact previously reported for the human class III aldehyde dehydrogenase. In human liver homogenate (cytosolic + microsomal fraction), the ALDH activity is easily detectable using either MONAL-71 or MONAL-62, with specific activities of approximately 2.5 and 3.2 units per gram of protein, respectively. The low apparent Km values, 0.85 and < 0.03 microM, respectively, together with the inhibition profile by propionic aldehyde (ID50 in the micromolar range) indicate that both compounds are oxidized primarily by the class I ALDH, further confirmed by low activity (0.4 U/g) with NADP+ as oxidant. By contrast, in human stomach, containing mostly class III ALDH, the activity measured with MONAL-71, 0.4 U/g, is much lower than that with MONAL-62 (5.1 U/g with NAD+ and 3.1 U/g with NADP+), the latter being virtually insensitive to 1 mM propionic aldehyde. Hence, in a stomach homogenate, class I and class III ALDH activities can be measured selectively with the two fluorogenic substrates described. In all experiments, the activity of aldehyde oxidase was at least 10-fold lower than that of the ALDH. Addition of 5 mM 4-methylpyrazole, a known inhibitor of the alcohol dehydrogenase, did not change the resultant ALDH activities by more than 10%, indicating lack of interference by the former enzyme. A preliminary screening of two liver tumour samples showed diminished class I ALDH activities (0.7 and 0.03 U/g), but no evidence for class III ALDH induction. The above observations are discussed in relation to the mechanism of detoxication of cyclophosphamide.
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PMID:Fluorimetric detection of aldehyde dehydrogenase activity in human blood, saliva, and organ biopsies and kinetic differentiation between class I and class III isozymes. 902 70

The metabolism of acetaldehyde (ACA), benzaldehyde (BA), propionaldehyde (PA) and valeraldehyde (VA) has been studied in two hepatoma cell lines, the rat HTC and mouse Hepa 1c1c7 cells. The cytotoxicity of the four aldehydes to these two cell lines has been compared. The end-points for evaluating cytotoxicity were 1) total macromolecular content (TMC) of confluent cultures, and 2) colony forming ability of dividing cells. These two assay systems had different sensitivities for the toxicity of aldehydes, probably due to different numbers of target cells. The activities of aldehyde dehydrogenases (NAD- and NADP-dependent, ALDH), alcohol dehydrogenase and aldehyde reductase were markedly greater in the HTC cell line compared to the Hepa 1c1c7 cell line, especially with BA as substrate. The cytotoxicities of aldehydes were generally stronger in the HTC cell line than in the Hepa 1c1c7 cell line; with the CF test. Particularly, BA was highly toxic to the HTC cells, which possessed the highest ALDH levels. Moreover, the treatment with (diethylamino)benzaldehyde, an ALDH inhibitor, completely abolished the toxicity of BA. Taken together, all these findings suggest that several cell lines expressing different aldehyde metabolizing activities could be used especially in the pre-screening phase to distinguish the metabolism-dependent cytotoxic effects from the metabolism independent effects.
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PMID:Comparative evaluation of cytotoxicity and metabolism of four aldehydes in two hepatoma cell lines. 929 76

The so far unelucidated pathway of formation of ethanol, one of the major end products of the fermentative metabolism of the amitochondriate protist, Giardia lamblia, was examined. Two NAD-dependent enzymatic activities, an acetaldehyde dehydrogenase (CoA-acetylating) (EC 1.2.1.10) and an alcohol dehydrogenase (EC 1.1.1.1) were detected. These are assumed to catalyze the formation of ethanol from acetyl-CoA via acetaldehyde. The first activity, present on a 95-kDa protein, was purified. It catalyzed the reversible interconversion of acetyl-CoA to acetaldehyde and CoA-SH with NAD but not NADP as cofactor. In the direction of aldehyde formation acetyl-CoA was the preferred substrate. Propionyl-CoA and isobutyryl-CoA were reduced with lower efficiency while succinyl-CoA and benzoyl-CoA were not. In the direction of acyl-CoA formation, acetaldehyde was the preferred substrate. Propionaldehyde and isobutyraldehyde were utilized at a lower efficiency while formaldehyde, benzaldehyde, and acetone were not. The second activity, a primary alcohol dehydrogenase, was also NAD-specific and used preferentially ethanol as substrate. Sequencing data of peptides from the purified protein and Northern and Southern analysis indicated that the same polypeptide, which belongs to the bifunctional aldehyde/alcohol dehydrogenase enzyme family, carried both activities. These activities define the pathway to ethanol in G. lamblia as a two step-processes: (i) acetyl-CoA + NADH<-->acetaldehyde + CoA-SH + NAD+ and (ii) acetaldehyde + NADH<-->ethanol + NAD+. In contrast to most eukaryotes in which ethanol formation proceeds from pyruvate via acetaldehyde, the G. lamblia pathway departs from acetyl-CoA, a more distal product of extended glycolysis.
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PMID:Aldehyde dehydrogenase (CoA-acetylating) and the mechanism of ethanol formation in the amitochondriate protist, Giardia lamblia. 963 98

Various strategies based on the use of chemically modified electrodes for the development of amperometric biosensors are described. Particular emphasis is placed on materials capable of catalyzing the oxidation of NADH and coupling these with enzymatic activities for biosensor construction. In terms of electrocatalysts, the discussion will centre on electrodeposited films of 3,4-dihydroxy benzaldehyde (3,4-DHB) and related analogs as well as on electrodeposited films of transition metal complexes of 1,10-phenanthroline-5,6-dione (phen-dione). Electrodeposited films of these materials have been coupled to the enzymatic activity of aldehyde dehydrogenase and alcohol dehydrogenase for the development of biosensors for aldehydes and ethanol, respectively.
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PMID:Analytical strategies for amperometric biosensors based on chemically modified electrodes. 964 68

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 &mgr;M for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.
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PMID:Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from eutypa lata1 995 58

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100-1000-fold less efficiently. The best kcat/Km values were found with benzaldehyde > 3-methoxybenzaldehyde > octanal > coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.
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PMID:Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase. 1033 11

The participation of Val-292 in catalysis by alcohol dehydrogenase and the involvement of dynamics were investigated. Val-292 interacts with the nicotinamide ring of the bound coenzyme and may facilitate hydride transfer. The substitution of Val-292 with Ser (V292S) increases the dissociation constants for the coenzymes (NAD(+) by 50-fold, NADH by 75-fold) and the turnover numbers by 3-7-fold. The V292S enzyme crystallized in the presence of NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol has an open conformation similar to the structure of the wild-type apo-enzyme, rather than the closed conformation observed for ternary complexes with wild-type enzyme. The V292S substitution perturbs the conformational equilibrium of the enzyme and decreases the kinetic complexity, which permits study of the hydride transfer step with steady-state kinetics. Eyring plots show that the DeltaH for the oxidation (V(1)) of the protio and deuterio benzyl alcohols is 13 kcal/mol and that the kinetic isotope effect of 4.1 is essentially temperature-independent. Eyring plots for the catalytic efficiency for reduction of benzaldehyde (V(2)/K(p)) with NADH or NADD are distinctly convex, being temperature-dependent from 5 to 25 degrees C and temperature-independent from 25 to 50 degrees C; the kinetic isotope effect of 3.2 for V(2)/K(p) is essentially independent of the temperature. The temperature dependencies and isotope effects for V(1) and V(2)/K(p) are not adequately explained by semiclassical transition state theory and are better explained by hydride transfer occurring through vibrationally assisted tunneling.
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PMID:Contributions of valine-292 in the nicotinamide binding site of liver alcohol dehydrogenase and dynamics to catalysis. 1160 93


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