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.     

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

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.     

Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase.

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.     

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

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.     

Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are 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.     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.     

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.     

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.     

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.     

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

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.     

Evidence for two distinct active sites on aldehyde dehydrogenase

Aldehyde dehydrogenase can catalyze the hydrolysis of esters such as p-nitrophenyl acetate as well as oxidize aldehydes to acids. It has not been proven unequivocally that the two reactions occur at the same active site. In the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1212-1217) evidence was presented which showed that cysteine at position 49 was at the active site for the dehydrogenase reaction. Evidence also was presented which showed that cysteine located at position 162 was susceptible to modification by N-ethylmaleimide. It was shown here that the two activities of the enzyme can be differently protected from inactivation by substrate analogs. Furthermore, aldehydes were found to be poor inhibitors against the esterase reaction while ester was a good inhibitor against the dehydrogenase reaction. In addition, it was possible to modify cysteine 49 with N-ethylmaleimide but not find inhibition of the esterase reactivity until cysteine 162 was modified. It appears that horse liver aldehyde dehydrogenase has two separate active sites per subunit. The data fit a model where ester can be hydrolyzed at both sites but that aldehyde oxidation occurred only at position 49.     

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.     

Structure elucidation of two differentiation inducing factors (DIF-2 and DIF-3) from the cellular slime mould Dictyostelium discoideum.

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.     

Alanine dehydrogenase of the N2-fixing blue-green alga, Anabaena cylindrica.

The L-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270,000, has 6 sub-units, each of molecular weight approximately 43,000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD+ specific and oxaloacetate can partially substitute for pyruvate. The Kampp for NAD+ is 14 muM and 60 muM at low and high NAD concentrations respectively.     

UDPglucose dehydrogenase. Kinetics and their mechanistic implications.

Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.     

The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.     

Effects of diethylstilbestrol, 2,2'-dithiodipyridine, and chloral hydrate on the esterase activity of sheep liver cytoplasmic aldehyde dehydrogenase

The binding of diethylstilbestrol (DES) to aldehyde dehydrogenase (ALDH) has a very similar effect on the dehydrogenase activity of the enzyme as has modification of the enzyme by 2,2'-dithiodipyridine [Kitson, T.M. (1982) Biochem. J. 207, 81-89]. The latter modification may occur at the site of the esterase activity of the enzyme [Kitson, T.M. (1985) Biochem. J. 228, 765-767]. This suggests that DES might be a competitive inhibitor of the esterase reaction. However, in the absence of oxidized nicotinamide adenine dinucleotide (NAD+) or reduced nicotinamide adenine dinucleotide (NADH), and at low concentrations of substrate (4-nitrophenyl acetate, PNPA), DES is a potent partial noncompetitive inhibitor. It is concluded therefore that DES binds at a site different from the esterase active site and that the enzyme-DES complex retains some ability to act as an esterase. High concentrations of PNPA appear to displace DES from its binding site. In the presence of NAD+, DES is a weaker inhibitor, and in the presence of NADH, DES has very little effect. Esterase activity is enhanced by NADH when PNPA concentrations are high but is inhibited when they are low. The rate of reaction of ALDH with 2,2'-dithiodipyridine is only slightly reduced by DES, suggesting that the site at which thiol modifiers react and the DES binding site are different. When ALDH is modified by 2,2'-dithiodipyridine, it has reduced esterase activity, which declines further as the modified enzyme loses its 2-thiopyridyl label. In the presence of NAD+, chloral hydrate is a simple competitive inhibitor of the esterase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Chemical studies of high-Km aldehyde dehydrogenase from rat liver mitochondria

Studies of pH-dependent kinetics implicate two ionizable groups in the dehydrogenase and esterase reactions catalysed by high-Km aldehyde dehydrogenase from rat liver mitochondria. Sensitized photooxidation completely arrests the bifunctional activities of the dehydrogenase. Carboxamidomethylation abolishes the dehydrogenase activity, whereas acetimidination eliminates the esterase activity. These results suggest that histidine (pKa near 6) and cysteine (pKa near 10) are likely the catalytic residues for the dehydrogenase activity, while the esterase activity is functionally related to histidine (pKa near 7) and a residue with the pKa value of 10-11. The two residues, a carboxyl group and an arginine, that discriminate between NAD+ and NADP+ are present at the coenzyme binding site of the mitochondrial high-Km aldehyde dehydrogenase from rat liver.