Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free 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.     

Subcellular distribution and properties of aldehyde dehydrogenase in the rat liver

The subcellular distribution and certain properties of rat liver aldehyde dehydrogenase are investigated. The enzyme is shown to be localized in fractions of mitochondria and microsomes. Optimal conditions are chosen for detecting the aldehyde dehydrogenase activity in the mentioned fractions. The enzyme of mitochondrial fraction shows the activity at low (0,03-0.05 mM; isoenzyme I) and high (5 mM; isoenzyme II) concentrations of the substrate. The seeming Km and V of aldehyde dehydrogenase from fractions of mitochondria and microsomes of rat liver are calculated, the acetaldehyde and NAD+ reaction being used as a substrate.     

Progress in establishing the rate-limiting features and kinetic mechanism of the glyceraldehyde-3-phosphate dehydrogenase reaction.

Primary hydrogen isotope effects and steady-state kinetics have been used to study the mechanism of glyceraldehyde-3-phosphate (GAP) dehydrogenase at pH 8.6. The isotope effect determined by using GAP-1d was unity and independent of arsenate (used as the acyl acceptor) and NAD+ concentrations when the aldehyde substrate was at saturating concentrations. At low GAP concentrations (apparent V/K conditions), the primary hydrogen isotope effect (H/D) was in the range of 1.40-1.52 and independent of arsenate and NAD+ concentrations. Apparent V/K for NAD+ was independent of GAP concentration, and apparent V/K for GAP was independent of NAD+ concentration. The dependence of apparent V/K for GAP on arsenate concentration was more complex but extrapolated to nonzero V/K at the zero-arsenate intercept. These observations are consistent with the general features of the Segal and Boyer (1953) mechanism for the reaction.     

Structure and mechanism of human UDP-glucose 6-dehydrogenase

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD(+)-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD(+) binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD(+) binding domain.     

The effect of quercetin, a widely distributed flavonoid in food and drink, on cytosolic aldehyde dehydrogenase: a comparison with the effect of diethylstilboestrol

Quercetin is a flavonoid found in red wine and many other dietary sources. Observations concerning the state of ionisation and the stability of the compound over a range of pH are presented. Quercetin is a potent inhibitor of cytosolic aldehyde dehydrogenase at physiological pH when the concentration of either the substrate or the cofactor is relatively low, but it has an activatory effect when the concentrations of substrate and cofactor are both high (1 mM). Gel filtration experiments show that quercetin binds very tightly to the enzyme under conditions where the compound is neutral and when it is ionised. The binding is less in the presence of NAD(+). Quercetin cuts down the ability of the resorufin anion to bind to the enzyme. The observations are explained by a model in which quercetin binds competitively to both the coenzyme-binding site and the aldehyde-binding site; binding in the latter location, when the enzyme is in the form of the E-NADH complex, accounts for the activation. The effects of quercetin are significantly different in some respects from those of diethylstilboestrol; this is explained by the latter being able to bind to the aldehyde site but not the NAD(+) site. The possibility that quercetin may affect aldehyde dehydrogenase in vivo is discussed.     

A mitochondrial NADP+-dependent reductase related to the 4-aminobutyrate shunt. Purification, characterization, and mechanism

Succinic semialdehyde reductase, a NADP+-dependent enzyme, was purified from whole pig brain homogenates. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Succinic semialdehyde reductase (Mr 110,000) catalyzes the reduction of succinic semialdehyde to 4-hydroxybutyrate. The equilibrium constant of the reaction is Keq = 5.8 X 10(7) M-1 at pH 7 and 25 degrees C. The inhibition kinetic patterns obtained when 4-hydroxybutyrate or substrate analogs are used as inhibitors of the reaction catalyzed by the reductase are consistent with an ordered sequential mechanism, in which the coenzyme NADPH adds to the enzyme before the aldehyde substrate. A specific aldehyde reductase was also purified to homogeneity from brain mitochondria preparations. Its catalytic properties are identical to those of the enzyme isolated from whole brain homogenates. It is postulated that two enzymes, i.e. a NAD+-dependent dehydrogenase and a NADP+-dependent reductase, participate in the metabolism of succinic semialdehyde in the mitochondria matrix.     

The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.     

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.     

Physiological role of yeasts NAD(P)+ and NADP+-linked aldehyde dehydrogenases.

The activity of NAD+ and NADP+-linked aldehyde dehydrogenases has been investigated in yeast cells grown under different conditions. As occurs in other dehydrogenase reactions the NAD(P)+-linked enzyme was strongly repressed in all hypoxic conditions; nervetheless, the NADP+-linked enzyme was active. The results suggest that the NAD(P)+ aldehyde dehydrogenase is involved in the oxidation of ethanol to acetyl-CoA, and that when the pyruvate dehydrogenase complex is repressed the NADP+-linked aldehyde dehydrogenase is operative as an alternative pathway from pyruvate to acetyl-CoA: pyruvate leads to acetaldehyde leads to acetate leads to acetyl-Coa. In these conditions the supply of NADPH is advantageous to the cellular economy for biosynthetic purposes. Short term adaptation experiments suggest that the regulation of the levels of the aldehyde dehydrogenase-NAD(P)+ takes place by the de novo synthesis of the enzyme.     

19F nuclear magnetic resonance observations of aldehyde dismutation catalyzed by horse liver alcohol dehydrogenase

We have examined aspects of the second catalytic activity of alcohol dehydrogenase from horse liver (LADH), which involves an apparent dismutation of an aldehyde substrate into alcohol and acid in the presence of LADH and NAD. Using the substrate p-trifluoromethylbenzaldehyde, we have observed various bound complexes by ¹⁹F NMR in an effort to further characterize the mechanism of the reaction. The mechanism appears to involve the catalytic activity of LADH · NAD · aldehyde complex which reacts to form an enzyme · NADH · acid complex. The affinity of the acid product for LADH · NADH is weak and the acid product readily desorbs from the ternary complex. The resulting LADH · NADH can then react with a second molecule of aldehyde to form NAD and the corresponding alcohol. The result is the conversion of two molecules of aldehyde to one each of acid and alcohol, with LADH and NAD acting catalytically. This sequence of reactions can also explain the slow formation of acid product observed when alcohol and NAD are incubated with the enzyme.     

Complete kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.     

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.     

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase. The effect of pH on the aldehyde binding reactions and a re-examination of the problem of the site of proton release in the mechanism.

Initial-rate measurements and stopped-flow spectrophotometric experiments over a wide range of pH implicate an enzyme group of pKa approximately 6.6 affecting the aldehyde binding reactions. It is possible, though not proved, that the group involved is the cysteine residue involved in catalysis. Stopped-flow fluorescence studies show that a group of pKa greater than 8.5 facilitates hydrolysis of the NADH-containing acyl-enzyme species. The identity of this group is quite unknown. Studies with 4-nitrobenzaldehyde show that this substrate gives marked substrate inhibition at quite low (less than 20 microM) concentrations. The mechanism of catalysis seems to be the same as for propionaldehyde oxidation. It is argued that proton release occurs with both substrates on hydrolysis of the NADH-containing acyl-enzyme and not before hydride transfer, as has been previously suggested [Bennett, Buckley & Blackwell (1982) Biochemistry 21, 4407-4413].     

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase.

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.     

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.     

Studies on pig muscle aldose reductase. Kinetic mechanism and evidence for a slow conformational change upon coenzyme binding.

Steady state kinetic analysis at pH 7.0 of the reduction of DL-glyceraldehyde by pig muscle aldose reductase showed that the enzyme follows a sequential ordered mechanism with NADPH binding first. However, the "off constant" for NADP+ in the forward direction was 1 order of magnitude less than the kcat. Analysis of this anomaly by pre-steady state kinetics using stopped-flow fluorescence spectroscopy showed that this could be accounted for by isomerization of the enzyme-NADP+ complex and that the rate of isomerization is the rate-limiting step. The rate constant for this step was of the same order of magnitude as the kcat for the forward reaction. Fluorescence emission spectra of free and NADP(H)-bound enzyme suggested a conformational change upon binding of coenzyme. In the reverse direction (oxidation of glycerol) pre-steady state and steady state kinetic analyses were consistent with the rate-limiting step occurring before isomerization of the enzyme-NADPH complex. We conclude, therefore, that during the kinetic mechanism of the reduction of aldehydes by aldose reductase, a slow (kinetically detectable) conformational change in the enzyme occurs upon coenzyme binding. Since NADPH and NADP+ bind to the enzyme very tightly, this has implications for the targeting and binding of drugs that are aldose reductase inhibitors.     

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.     

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.     

Inhibition of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.