Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.     

Human liver akdehyde dehydrogenase. Kinetics of aldehyde oxidation.

Steady state initial velocity studies were carried out to determine the kinetic mechanism of human liver aldehyde dehydrogenase. Intersecting double reciprocal plots obtained in the absence of inhibitors demonstrated that the dehydrogenase reaction proceeded by sequential addition of both substrates prior to release of products. Dead end inhibition patterns obtained with coenzyme and substrate analogues (e.g. thionicotinamide-AD+ and chloral hydrate) indicated that NAD+ and aldehyde can bind in random fashion. The patterns of inhibition by the product NADH and of substrate inhibition by glyceraldehyde were also consistent with this mechanism. However, comparisons between kinetic constants associated with the dehydrogenase and esterase activities of this enzyme suggested that most of the dehydrogenase reaction flux proceeds via formation of an initial binary NAD+-enzyme complex over a wide range of substrate and coenzyme concentrations.     

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.     

Characterization of rat cornea aldehyde dehydrogenase

Aldehyde dehydrogenase has been purified from rat cornea in a single step. The enzyme is a class 3 aldehyde dehydrogenase. Cornea aldehyde dehydrogenase is a 100-kDa dimer composed of 51-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes as well as medium chain length (4-9 carbons) aliphatic aldehydes. The substrate and coenzyme specificity, immunochemical properties, effect of disulfiram, pH profile, and isoelectric point of cornea aldehyde dehydrogenase are identical to those of tumor-associated aldehyde dehydrogenase, the prototype class 3 enzyme. The substrate and coenzyme preferences are consistent with a role for cornea aldehyde dehydrogenase in the oxidation of a variety of aldehydes generated by lipid metabolism, including lipid peroxidation.     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.     

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K(i)=47 microM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane-carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.     

Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme.

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 X 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.     

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K_i=47 μM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane–carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.     

Pyrroline 5-carboxylate dehydrogenase of the mitochondrial matrix of rat liver. Purification, physical and kinetic characteristics

The oxidation of proline to glutamate in mitochondria requires two enzymes, proline oxidase and pyrroline 5-carboxylate (P5C) dehydrogenase. In this paper we report an 800-fold purification P5C dehydrogenase from rat liver mitochondria to yield an essentially homogenous protein. The protein, whose Mr is 59,000, is an alpha 2 dimer (Mr = 115,000) in solution with an isoionic point at pH 5.7. The substrates P5C and NAD+ have apparent dissociation constants of 0.16 and 1.0 mM, respectively. Studies have been conducted to see if the conversion of glutamate and NADH to P5C and NAD+ is catalyzed by this enzyme. These studies have established that if the reverse reaction occurs the rate is 1/15,000th of the rate at which P5C is oxidized to glutamate. The concentration of the substrates needed in the assay results in a high background that interferes with accurate spectrophotometric analysis of the rate of NADH production; therefore a radiochemical (2) or a new colorimetric (3) assay was used here. A number of aldehydes were tested as substrates. It was found that the rat and human enzymes (4) have similar requirements for an aldehyde to be a substrate. Both of these proteins interacted with a polyclonal rabbit anti-rat P5C dehydrogenase serum.     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Purification and characterization of an NADH-linked aldehyde reductase from bovine brain

Abstract— The presence of a nonspecific NADH-linked aldehyde reductase was demonstrated in various regions of bovine brain in vitro. With m-nitrobenzaldehyde as substrate, the rate of NADH oxidation was approximately 4 nmol.min^-1.(mg of protein)^-1 in the cerebellum, pons and medulla; but somewhat lower rates [2–3 nmol.min^-1.(mg of protein)-^l] were obtained in the other areas of the brain examined. The enzyme was localized primarily in the soluble, supernatant fraction of rat brain homogenates. The enzyme from the supernatant fluid fraction of bovine brain was purified approximately 350-fold by ammonium sulphate fractionation and chromatography on calcium phosphate-gel, DEAE-cellulose and Sephadex G200 columns. The partially purified enzyme catalysed the reduction of a number of aldehydes, including substituted benzaldehydes and aliphatic aldehydes of intermediate chain lengths. Short chain aliphatic aldehydes, such as acetaldehyde, were not reduced by the enzyme and butyraldehyde was a poor substrate. With m-nitrobenzaldehyde as substrate, NADH was oxidized at an approximately 10-fold faster rate than NADPH. The pH optimum for the enzyme was 6.75 for aldehyde reduction, whereas the rate of oxidation of m-nitrobenzylalcohol was optimal at pH 10.0 with NAD as the co-substrate. K_m and K₃ values ranged from 10 μM to 10 mM for various aldehydes and from 10 to 30 μM for the cofactors. Oxidation of NADH by the partially purified enzyme was not inhibited by 10m pyrazole or by 1 mM phenobarbital. However, the enzyme activity was inhibited by approximately 60 percent by 1 mM chlorpromazine or by 5 mM 1,10-orthophenanthroline. Our data demonstrate that the enzyme is not only separable from the NADPH-linked aldehyde reductase described previously by TABAKOFF and ERWIN, but also is quite different in substrate specificity and inhibitor sensitivity from the ‘classical’, pyrazole-sensitive, NAD- linked alcohol dehydrogenase (EC 1.1.1.1).     

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli. D-mannitol-1-phosphate dehydrogenase was purified to homogeneity from Escherichia coli, and its physicochemical and enzymatic properties were investigated. The molecular weight of the polypeptide chain is 45,000 as determined by polyacrylamide gel electrophoresis in denaturing conditions. High performance size exclusion chromatography gives an apparent molecular weight of 47,000 for the native enzyme, showing that D-mannitol-1-phosphate dehydrogenase is a monomeric NAD-dependent dehydrogenase. D-mannitol-1-phosphate dehydrogenase is rapidly denatured by 6 M guanidine hydrochloride. Non-superimposable transition curves for the loss of activity and the changes in fluorescence suggest the existence of a partially folded inactive intermediate. The protein can be fully renatured after complete unfolding, and the regain of both native fluorescence and activity occurs rapidly within a few seconds at pH 7.5 and 20 degrees C. Such a high rate of reactivation is unusual for a protein of this size. D-mannitol-1-phosphate dehydrogenase is specific for mannitol-1-phosphate (or fructose-6-phosphate) as a substrate and NAD+ (or NADH) as a cofactor. Zinc is not required for the activity. The affinity of D-mannitol-1-phosphate dehydrogenase for the reduced or oxidized form of its substrate or cofactor remains constant with pH. The affinity for NADH is 20-fold higher than for NAD+. The forward and reverse catalytic rate constants of the reaction: mannitol-1-phosphate + NAD+ in equilibrium fructose-6-phosphate + NADH have different pH dependences. The oxidation of mannitol-1-phosphate has an optimum pH of 9.5, while the reduction of fructose-6-phosphate has its maximum rate at pH 7.0. At pH values around neutrality the maximum rate of reduction of fructose-6-phosphate is much higher than that of oxidation of mannitol-1-phosphate. The enzymatic properties of isolated D-mannitol-1-phosphate dehydrogenase are discussed in relation to the role of this enzyme in the intracellular metabolism.     

Characterisation of recombinant human fatty aldehyde dehydrogenase: implications for Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sj?gren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of approximately 9.5 and temperature of approximately 35 degrees C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25-30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.     

NAD(P)+-independent aldehyde dehydrogenase from Pseudomonas testosteroni. A novel type of molybdenum-containing hydroxylase

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6-dichloroindophenol), NAD(P)+ or O2, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186,000, consists of two subunits of equal size (Mr 92,000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+-independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde-converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quino-protein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases.     

Functional characterization of a Drosophila melanogaster succinic semialdehyde dehydrogenase and a non-specific aldehyde dehydrogenase

The putative Drosophila (D.) melanogaster gene ortholog of mammalian succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24; NM_143151) that is involved in the degradation of the neurotransmitter GABA, and the putative D. melanogaster aldehyde dehydrogenase gene Aldh (NM_135441) were cloned and expressed as enzymatically active maltose binding protein (MalE) fusion products in Escherichia coli. The identities of the NM_143151 gene product as NAD+-dependent SSADH and of the Aldh gene product as NAD+-dependent non-specific aldehyde dehydrogenase (ALDH, EC1.2.1.3) were established by substrate specificity studies using 30 different aldehydes. In the case of D. melanogaster MalE-SSADH, the Michaelis constants (K(M)s) for the specific substrates succinic semialdehyde and NAD+ was 4.7 and 90.9 microM, respectively. For D. melanogaster MalE-ALDH the K(M) of the putative in vivo substrate acetaldehyde was 0.9 microM while for NAD+, a K(M) of 62.7 microM was determined. Site-directed mutagenesis studies on D. melanogaster MalE-SSADH suggest that cysteine 311 and glutamic acid 277 of this enzyme are likely candidates for the active site residues directly involved in catalysis.     

Substrate Specificity, Substrate Channeling, and Allostery in BphJ: An Acylating Aldehyde Dehydrogenase Associated with the Pyruvate Aldolase BphI

BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling.     

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin, resveratrol and diethylstilbestrol

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD(+)-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD(+) concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD(+), by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.     

Reversal of part of the aldehyde dehydrogenase reaction pathway during the hydrolysis of an ester.

An aldehyde dehydrogenase from rabbit liver, a homogeneous protein on three distinct polyacrylamide-gel systems, has an associated 4-nitrophenyl esterase activity. At pH 7.0 in the presence of 80 micrometer-NADH and 800 micrometer-4-nitrophenyl acetate the enzyme produces NAD+ and a stoicheiometric amount of an aldehyde, as well as hydrolysing the ester. On this and other evidence it is proposed that ester hydrolysis occurs at the usual active site of the enzyme.