Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes.

Human liver extracts show two major bands with aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) activity via starch gel electrophoresis at pH 7.0. Both bands have been purified to apparent homogeneity via classical chromatography combined with affinity chromatography on 5'-AMP-Sepharose 4B. The slower migrating band, enzyme 1, when assayed at pH 9.5 has a low Km for NAD (8 micrometer) and a high Km for acetaldehyde (approx. 0.1 mM). It is very strongly inhibited by disulfiram at pH 7.0 with a Ki of 0.2 micrometer. The faster migrating band, enzyme 2, has a low Km for acetaldehyde, (2--3 micrometer at pH 9.5), a higher Km for NAD (70 micrometer at pH 9.5), and is not inhibited by disulfiram at pH 7.0. The two enzymes are very similar to the F1 and F2 isozymes of horse liver purified by Eckfeldt et al. (Eckfeldt, J., Mope, L., Takio, K. and Yonetani, T. (1976) J. Biol, Chem. 251, 236-240) in molecular weight, subunit composition, amino acid composition and extinction coefficient. Preliminary kinetic characterizations of the enzyme are presented.     

Metabolism of acetaldehyde by human erythrocytes.

Acetaldehyde metabolism in human erythrocytes was studied using head-space gas chromatographic determination methods, and it was found that acetaldehyde is metabolized in erythrocytes by NAD dependent cytosolic enzyme having an apparent Km value for acetaldehyde approximately 0.7 mM at pH 7.4, and more than 50% of this activity was reduced by 1 μM disulfiram. So, it is suggested that erythrocytes may have an enzyme system similar to the high Km isozyme of the liver aldehyde dehydrogenase.     

Liver alcohol dehydrogenase and aldehyde dehydrogenase in the Japanese: isozyme variation and its possible role in alcohol intoxication.

Forty autopsy livers from Japanese individuals were studied concerning alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes using electrophoretic and enzyme assay methods. A remarkably high frequency (85%) was found for the atypical ADH phenotype. The gene frequencies of ADH22 and ADH32 were .625 and .05, respectively. The usual ALDH phenotype showed two major isozyme bands, a faster migrating (low Km for acetaldehyde) and a slower migrating isozyme (high Km for acetaldehyde). Fifty-two percent of the specimens had an unusual phenotype of ALDH, which showed only the slower migrating isozyme. The usual phenotype was inhibited about 20%--30% by disulfiram and the unusual type up to 90%. Such a high incidence in the Japanese of the unusual phenotype, which lacks in the low Km isozyme, suggests that the initial intoxicating symptoms after alcohol drinking in these subjects might be due to delayed oxidation of acetaldehyde rather than its higher-than-normal production by typical or atypical ADH.     

Subcellular localization of the F1 and F2 isozymes of horse liver aldehyde dehydrogenase

The subcellular distribution and relative amounts of the two isozymes, F1 and F2, of aldehyde dehydrogenase (EC 1.2.1.3) which were recently purified to homogeneity from horse liver (Eckfeldt, J., et al. (1976) J. Biol. Chem.251, 236–240) have been investigated. A fresh horse liver homogenate was fractionated on DEAE-cellulose. The results indicate that approximately 60% of the total pH 7.0 acetaldehyde dehydrogenase activity is due to the F1 isozyme and 40% is due to the F2 isozyme. Several horse livers were then fractionated into subcellular components using a differential centrifugation method. Based on the disulfiram (Antabuse) inhibition and the aldehyde concentration dependence of the enzymatic activity, it appears that the disulfiram-sensitive F1 isozyme (K_m acetaldehyde ? 70 μm) is primarily cytosolic and the disulfiram-insensitive F2 isozyme (K_{m acetaldehyde} ? 0.2 μm) is primarily mitochondrial. Fluorescence studies showed that the acetaldehyde dehydrogenase of the intact mitochondria could utilize only the endogenous pyridine nucleotide pool and not externally added NAD. Also, the ethanol dehydrogenase activity was found to be nearly 10 times the total acetaldehyde dehydrogenase activity when assaying a horse liver homogenate at pH 7.0 and with saturating substrates. The significant differences between this work and the results reported in rat liver are discussed with respect to the physiological importance of the cytosolic and mitochondrial aldehyde dehydrogenase during the ethanol oxidation in vivo.     

Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.     

Human aldehyde dehydrogenase. Activity with aldehyde metabolites of monoamines, diamines, and polyamines

Two isozymes (E1 and E2) of human aldehyde dehydrogenase (EC 1.2.1.3) were purified to homogeneity 13 years ago and a third isozyme (E3) with a low Km for gamma-aminobutyraldehyde only recently. Comparison with a variety of substrates demonstrates that substrate specificity of all three isozymes is broad and similar. With straight chain aliphatic aldehydes (C1-C6) the Km values of the E3 isozyme are identical with those of the E1 isozyme. All isozymes dehydrogenate naturally occurring aldehydes, 5-imidazoleacetaldehyde (histamine metabolite) and acrolein (product of beta-elimination of oxidized polyamines) with similar catalytic efficiency. Differences between the isozymes are in the Km values for aminoaldehydes. Although all isozymes can dehydrogenate gamma-aminobutyraldehyde, the Km value of the E3 isozyme is much lower: the same appears to apply to aldehyde metabolites of cadaverine, agmatine, spermidine, and spermine for which Km values range between 2-18 microM and kcat values between 0.8-1.9 mumol/min/mg. Thus, the E3 isozyme has properties which make it suitable for the metabolism of aminoaldehydes. The physiological role of E1 and E2 isozymes could be in dehydrogenation of aldehyde metabolites of monoamines such as 3,4-dihydroxyphenylacetaldehyde or 5-hydroxyindoleacetaldehyde; the catalytic efficiency with these substrates is better with E1 and E2 isozymes than with E3 isozyme. Isoelectric focusing of liver homogenates followed by development with various physiological substrates together with substrate specificity data suggest that aldehyde dehydrogenase (EC 1.2.1.3) is the only enzyme in the human liver capable of catalyzing dehydrogenation of aldehydes arising via monoamine, diamine, and plasma amine oxidases. Although the enzyme is generally considered to function in detoxication, our data suggest an additional function in metabolism of biogenic amines.     

Structural mutation in a major human aldehyde dehydrogenase gene results in loss of enzyme activity.

Most Caucasians have two major liver aldehyde dehydrogenase isozymes, ALDH1 and ALDH2, while approximately 50% of Orientals have only ALDH1 isozyme, missing the ALDH2 isozyme. A remarkably higher frequency of acute alcohol intoxication among Orientals than among Caucasians could be related to the absence of the ALDH2 isozyme, which has a low apparent Km for acetaldehyde. Examination of liver extracts by two-dimensional crossed immunoelectrophoresis revealed that an atypical Japanese liver, which had no ALDH2 isozyme, contained an enzymatically inactive but immunologically cross-reactive material corresponding to ALDH2, beside the active ALDH1 isozyme. Therefore, the absence of ALDH2 isozyme in atypical Orientals is not due to regulatory mutation, gene deletion, or nonsense mutation, but must be due to a structural mutation in a gene for the ALDH2 locus, resulting in synthesis of enzymatically inactive abnormal protein.     

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.     

A chemical and enzymological account of the multiple forms of human liver aldehyde dehydrogenase. Implications for ethnic differences in alcohol metabolism

Three major low-pI zones of aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) may be visualized with specific histochemical staining after starch gel electrophoresis at pH 7.4 of Caucasian human liver extracts, whereas about 50% of Chinese human liver extracts show only two such zones. The three zones of activity were purified to apparent homogeneity from Caucasian liver. The substrate specificity of each form was investigated by double reciprocal plots using 13 aldehydes of various chemistries. The acetaldehyde-preferring isozyme I lacking in 50% of Chinese livers had a slightly lower native and subunit molecular weight than the "universal' isozymes IIa and IIb. All forms were highly sensitive to disulfiram inhibition. This inhibition could be protected against, or reversed, by dithiothreitol. 2,2'-Dithiodipyridine was a slower inhibitor of isoenzyme I. All three purified forms of the enzyme, as well as crude extracts of normal and isozyme I-deficient Chinese livers, showed positive immunoreactivity to antibodies prepared in rabbits against type I enzyme. Tryptic peptide maps of forms IIa and IIb were almost identical, whereas that of form I, although showing some similarities, was clearly different. These results provide a consistent explanation for the acetaldehyde-mediated extreme sensitivity to moderate alcohol ingestion shown normally by about 50% of oriental subjects and during disulfiram (Antabuse) therapy by all subjects.     

Mouse mitochondrial aldehyde dehydrogenase isozymes: purification and molecular properties

Aldehyde dehydrogenase isozymes (AHD-1 and AHD-5) have been isolated in a highly purified state from extracts of mouse liver mitochondria. The enzymes have distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: AHD-1, 63,000; AHD-5, 49,000. Gel exclusion chromatography, using sephadex G-200, indicated that both isozymes are dimers, although AHD-1 may also exist as a monomeric form as well. The enzymes exhibited widely divergent kinetic characteristics. The purified allelic forms of AHD-1, AHD-1A (C57BL/6J mice) and AHD-1B (CBA/H mice), exhibited high Km values with acetaldehyde as substrate, 1.4 mM and 0.78 mM respectively, whereas AHD-5 exhibited a low Km value with acetaldehyde of 0.2 microM. In addition, the isozymes exhibited distinct pH optima for catalysis (AHD-1, pH range 6.5-7.5; AHD-5, pH range 8.5-10.0), and were differentially sensitive towards disulphuram inhibition, with 50% inhibition occurring 13 and 0.1 microM for the AHD-1 and AHD-5 isozyme respectively. Based upon the kinetic characteristics, it is suggested that AHD-5 may be the primary enzyme for oxidizing mitochondrial acetaldehyde during ethanol oxidation in vivo.     

Subcellular localization and capacity of beta-oxidation and aldehyde dehydrogenase in porcine liver

Porcine hepatocyte organelles were separated by isopycnic sucrose gradient centrifugation from livers of 6-month-old Yorkshire pigs. The presence of a peroxisomal palmitoyl-CoA oxidizing system and a peroxisomal NAD:aldehyde dehydrogenase (ALDH) with high Km for acetaldehyde was demonstrated. Peroxisomal palmitate oxidizing capacity was found to be equal to that of the surviving mitochondria. The high Km isozyme of ALDH was mainly located in the mitochondria (54%), with a significant portion in the peroxisome (32%). Remaining activity is distributed among the microsomes (8.3%) and cytosol (4.6%). The low Km isozyme was confined almost exclusively to the mitochondria. ALDH may exist in the peroxisome as a detoxification mechanism and contribute to shorter half-lives of reactive aldehydes in the cell. Species differences are discussed.     

Acetaldehyde oxidation in rat liver mitochondria, action of Mg2+, ATP and rotenone on acetaldehyde oxidation in intact mitochondria, and on some purified aldehyde dehydrogenase isozymes

In intact rat liver mitochondria acetaldehyde is oxidized by three functionally distinct dehydrogenase systems. Two of these reduce intramitochondrial nicotinamide adenine dinucleotide (NAD): one is operative with micromolar acetaldehyde concentrations and is stimulated by Mg2+, the other is operative with millimolar acetaldehyde concentrations and is stimulated by adenosine 5'-triphosphate (ATP). The third system reduces added NAD and is stimulated by rotenone. Connected to these systems, three aldehyde dehydrogenase isozymes (ALDH) have been purified: a low-Km ALDH activated by Mg2+, a high-Km ALDH activated by ATP and Mg2+, a high-Km ALDH activated by rotenone. The properties of some isozymes are affected by detergents. Thus, deoxycholate augments the stimulation of low-Km isozyme by Mg2+ and confers sensitivity to Mg2+ and ATP on one of the high-Km isozymes. A fourth isozyme has been purified. Its affinity for acetaldehyde is so low that it is very unlikely that acetaldehyde is the physiological substrate.     

Rat liver constitutive and phenobarbital-inducible cytosolic aldehyde dehydrogenases are highly homologous proteins that function as distinct isozymes

Rat liver contains two class 1 aldehyde dehydrogenases (ALDHs): a constitutive isozyme (ALDH1) and a phenobarbital-inducible isozyme (ALDH-PB). Defining characteristics of mammalian class 1 ALDHs include a homotetrameric structure, high expression in liver, sensitivity to the inhibitor disulfiram, and high activity for the oxidation of retinal. It is often presumed that ALDH-PB is the rat ortholog of mammalian ALDH1, and the identity of rat ALDH-PB is commonly interchanged with ALDH1. In this study, we characterized recombinant rat liver cytosolic ALDH1 and ALDH-PB. Previous reports indicate that ALDH-PB is a homodimer; however, we found by mass spectrometry and gel electrophoresis that it is a homotetramer. ALDH1 mRNA was highly expressed in untreated rat liver, while ALDH-PB had very weak expression, in contrast to a previous report that ALDH-PB mRNA is expressed in untreated rat liver. Rat liver ALDH1 had a high affinity for retinal (K(m) = 0.6 microM), while no oxidation by ALDH-PB could be detected with 20 microM retinal. ALDH1 was more efficient at oxidizing acetaldehyde, propionaldehyde, and benzaldehyde and was more sensitive to disulfiram inhibition. We conclude that rat liver ALDH1 is the ortholog of mammalian liver ALDH1. Furthermore, despite a high level of sequence identity and classification as a class 1 ALDH, ALDH-PB does not function like ALDH1. ALDH-PB is not merely an inducible ALDH1 isozyme; it is a distinct ALDH isozyme.     

Rat liver aldehyde dehydrogenase. I. Isolation and characterization of four high Km normal liver isozymes

From normal rat liver mitochondrial and microsomal fractions, 4 distinct aldehyde dehydrogenase isozymes with millimolar substrate Km values have been purified and characterized. Two isozymes were isolated from mitochondria and 2 from microsomes. A mitochondrial aldehyde dehydrogenase with a substrate Km in the micromolar range was also identified. Subunit molecular weights for all millimolar Km isozymes is 54,000. The mitochondrial and microsomal millimolar Km isozymes are clearly distinguishable from each other by substrate and coenzyme specificity, pH velocity profiles, and thermal stability. By these same properties, the 2 isozymes from each organelle are virtually identical. The 2 mitochondrial isozymes can be distinguished by apparent molecular weight (I, 170,000; II, approximately 250,000), Km for NADP+, effect of inhibitors, and pI. The 2 microsomal isozymes are of the same apparent molecular weight (approximately 250,000), but are distinguishable by their Km values for benzaldehyde and NADP+, response to inhibitors, and pI.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties.

1. Uronic acid dehydrogenase was purified to homogeneity. After a 338-fold purification a yield of 16% was achieved with a specific activity of 81 mumol NADH formed min-1 mg protein-1. 2. The purity of the enzyme was controlled by disc electrophoresis, sodium dodecylsulfate electrophoresis and ultracentrifugation. 3. A molecular weight of 60 000 was determined by gel chromatography and by ultracentrifugation. 4. The native enzyme is composed of two subunits, their molecular weight being 30 000 as estimated by sodium dodecylsulfate electrophoresis. The subunits as such are inactive. 5. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. 6. For catalytic activity no SH groups and no metals seem to be necessary. 7. The Michaelis constants determined with the pure enzyme are for glucuronic acid Km = 0.37 mM, galacturonic acid Km = 54 muM and NAD+ (with glucuronic acid) Km = 80 muM. 8. A weak reverse reaction could be observed with glucaric acid lactones at acidic pH. 9. NADH is competitive with NAD+. The inhibitor constant is Ki = 60 muM. 10. The NAD+ binding site seems to be of lower specificity than the uronic acid binding site.     

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.     

Human placental aldehyde dehydrogenase. Subcellular distribution and properties

Freshly obtained human term placentae were subjected to subcellular fractionation to study the localization of NAD-dependent aldehyde dehydrogenases. Optimal conditions for the cross-contamination-free subcellular fractionation were standardized as judged by the presence or the absence of appropriate marker enzymes. Two distinct isozymes, aldehyde dehydrogenase I and II, were detected in placental extracts after isoelectric focusing on polyacrylamide gels. Based on a placental wet weight, about 80% of the total aldehyde dehydrogenase activity was found in the cytosolic acid and about 10% in the mitochondrial fraction. The soluble fraction (cytosol) contained predominantly aldehyde dehydrogenase II which has a relatively high Km (9 mmol/l) for acetaldehyde and is strongly inhibited by disulfiram. The results indicate that cytosol is the main site for acetaldehyde oxidation, but the enzyme activity is too slow to prevent the placental passage of normal concentrations of blood acetaldehyde (less than 1 mumol/l) produced by maternal ethanol metabolism.     

The role of aldehyde dehydrogenases in the malonic dialdehyde metabolism in the rat liver

The enzymes catalyzing the NAD-dependent oxidation of malonic dialdehyde (MDA) were isolated from rat liver extracts. Upon 5'-AMP-Sepharose chromatography MDA dehydrogenase was separated into two isoforms, I and II. Isoform I was eluted from the affinity carrier with a 0.1 M phosphate buffer pH 8.0. This isoform had a broad substrate specificity towards aliphatic and aromatic aldehydes. Kinetic studies showed that short- and medium-chain aliphatic aldehydes (C2-C6) were characterized by the lowest Km values and the highest Vmax values. The Km' values for MDA and acetaldehyde were 2.8 microM and 0.69 microM, respectively. Isoform II was eluted with a 0.1 M phosphate buffer pH 8.0 containing 0.5 mM NAD, was the most active with medium- and long-chain aliphatic aldehydes (C6-C11) and had Km values for MDA and acetaldehyde equal to 37 microM and 52 microM, respectively. Isoform I was much more sensitive towards disulfiram inhibition than isoform II. Both isoforms had an identical molecular mass (93 kD) upon gel filtration. It is concluded that MDA dehydrogenase isoform I is identical to mitochondrial aldehyde dehydrogenase having a low Km for acetaldehyde, whereas isoform II may be localized in liver cytosol. The role of aldehyde dehydrogenases in the metabolism of aldehydes derived from lipid peroxidation is discussed.     

Degradation of vinyl acetate by soil, sewage, sludge, and the newly isolated aerobic bacterium V2.

Vinyl acetate is subject to microbial degradation in the environment and by pure cultures. It was hydrolyzed by samples of soil, sludge, and sewage at rates of up to 6.38 and 1 mmol/h per g (dry weight) under aerobic and anaerobic conditions, respectively. Four yeasts and thirteen bacteria that feed aerobically on vinyl acetate were isolated. The pathway of vinyl acetate degradation was studied in bacterium V2. Vinyl acetate was degraded to acetate as follows: vinyl acetate + NAD(P)+----2 acetate + NAD(P)H + H+. The acetate was then converted to acetyl coenzyme A and oxidized through the tricarboxylic acid cycle and the glyoxylate bypass. The key enzyme of the pathway is vinyl acetate esterase, which hydrolyzed the ester to acetate and vinyl alcohol. The latter isomerized spontaneously to acetaldehyde and was then converted to acetate. The acetaldehyde was disproportionated into ethanol and acetate. The enzymes involved in the metabolism of vinyl acetate were studied in extracts. Vinyl acetate esterase (Km = 6.13 mM) was also active with indoxyl acetate (Km = 0.98 mM), providing the basis for a convenient spectrophotometric test. Substrates of aldehyde dehydrogenase were formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde. The enzyme was equally active with NAD+ or NADP+. Alcohol dehydrogenase was active with ethanol (Km = 0.24 mM), 1-propanol (Km = 0.34 mM), and 1-butanol (Km = 0.16 mM) and was linked to NAD+. The molecular sizes of aldehyde dehydrogenase and alcohol dehydrogenase were 145 and 215 kilodaltons, respectively.