Kinetic mechanism of the hydroxypyruvate-lactate dehydrogenase-NADH system.

Chicken liver lactate dehydrogenase L-lactate : NAD+ oxidoreductase, EC1.1.1.27) reversibly catalyses the conversion of hydroxypyruvate to L-glycerate. The variation of the initial reaction rate with the substrate or coenzyme (NADH) concentration together with the inhibition caused by the reaction products and excess substrates, reveal that the kinetic mechanism of the reaction, with hydroxypyruvate as substrate, is of the rapid-equilibrium, ordered-ternary-complex type; NADH is the first substrate in the reaction sequence. Rate equations have been developed for the hydroxypyruvate.E.NADH system without inhibitors, with excess substrates, and with reaction products. Comparison of the rate equations obtained with those calculated theoretically from an ordered-ternary-complex mechanism reveals the existence of E.NAD.NADH,E.NAD-hydroxypyruvate and E.hydroxypyruvate complexes.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

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.     

Kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase

The kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase (EC 1.4.1.3) has been investigated at pH 9.0, 3 M NaCl and 40 degrees C in both directions, by initial rate and inhibition studies. The results of the initial rate studies indicate that the mechanism is sequential with respect to substrate addition. The inhibition patterns obtained with halophilic NAD+-glutamate dehydrogenase are not consistent with a simple ordered mechanism without modification. They can, however, be reconciled with this type of mechanism by postulating an appropriate abortive complex.     

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.     

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.     

A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase.

The kinetic mechanism of glutamate dehydrogenase with the monocarboxylic substrate norvaline was examined by using initial-rate steady-state kinetics and inhibition kinetics. To a first approximation the reaction mechanism can be described as a rapid-equilibrium random-order one. Binding synergism between the monocarboxylic substrate and coenzyme is not observed. Dissociation constants for NAD+ and 2-oxoglutarate calculated from the kinetic data assuming a rapid-equilibrium random-order model are in good agreement with independently obtained estimates. Lineweaver-Burk plots with varied norvaline concentration are not strictly linear, and it is concluded that a steady-state random-order model more accurately reflects the observed kinetics with norvaline as substrate.     

Kinetic study of porcine kidney betaine aldehyde dehydrogenase

Porcine kidney betaine aldehyde dehydrogenase (EC 1.2.1.8) kinetic properties were determined at low substrate concentrations. The double-reciprocal plots of initial velocity versus substrate concentration are linear and intersect at the left of the 1/v axis and showed substrate inhibition with betaine aldehyde. Studies of inhibition by NADH and dead-end analogs showed that NADH is a mixed inhibitor against NAD(+) and betaine aldehyde. AMP is competitive with respect to NAD(+) and mixed with betaine aldehyde. Choline is competitive against betaine aldehyde and uncompetitive with respect to NAD(+). The kinetic behavior is consistent with an Iso-Ordered Bi-Bi Steady-State mechanism.     

The reaction of pyruvate with saccharopine dehydrogenase.

The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxo-glutarate as a substrate of saccharopine dehydrogenase [epsilon-N-(L-glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine-forming) in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, L-lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2-oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+ was shown to be the last reactant released. Saccharopine [epsilon-N-(L-glutaryl-2)-L-lysine] was found to act as a potent dead-end inhibitor of the condensation reactions (of lysine and 2-oxoglutarate, and of lysine and pyruvate) by forming an abortive E. NADH. saccharopine complex.     

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.     

The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.     

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.     

A product-inhibition study of bovine liver glutamate dehydrogenase.

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.     

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.     

Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria

Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.     

Intra- and intermolecular electron transfer processes in redox proteins

Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition.     

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.     

Kinetic mechanism of chicken liver mitochondrial malatedehydrogenase (author's transl)]

Kinetic mechanism of chicken liver mitochondrial malatedehydrogenase is established from the initial reaction rates and reaction product inhibition. The results obtained in the reduction of NAD+ (pH 10) allow the formulation of an ordered bi-bi mechanism in which the coenzyme is the first substrate added to the enzyme. Inhibition by the reaction products and by the excess of the very substrates, reveals the formation of the E-oxaloacetate; E-L-malate; E-NAD+-oxaloacetate; E.NAD+-NADH; E-NADH-NADH and E-NAD-NAD+ abortive complexes, the significance of which may be checked under defined experimental conditions. Enzyme inhibition by ATP, ADP and AMP in the reduction of NAD+ (pH10), accords with the proposed mechanism and proceeds via formation of the E-nucleotide and E-NAD+-nucleotide inactive complexes.     

Drosophila alcohol dehydrogenase: acetate-enzyme interactions and novel insights into the effects of electrostatics on catalysis

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones and that is also able to further oxidize aldehydes to their corresponding carboxylic acids. The structure of the ternary enzyme-NADH-acetate complex of the slow alleloform of Drosophila melanogaster ADH (DmADH-S) was solved at 1.6 A resolution by X-ray crystallography. The coenzyme stereochemistry of the aldehyde dismutation reaction showed that the obtained enzyme-NADH-acetate complex reflects a productive ternary complex although no enzymatic reaction occurs. The stereochemistry of the acetate binding in the bifurcated substrate-binding site, along with previous stereochemical studies of aldehyde reduction and alcohol oxidation shows that the methyl group of the aldehyde in the reduction reaction binds to the R1 and in the oxidation reaction to the R2 sub-site. NMR studies along with previous kinetic studies show that the formed acetaldehyde intermediate in the oxidation of ethanol to acetate leaves the substrate site prior to the reduced coenzyme, and then binds to the newly formed enzyme-NAD+ complex. Here, we compare the three-dimensional structure of D.melanogaster ADH-S and a previous theoretically built model, evaluate the differences with the crystal structures of five Drosophila lebanonensis ADHs in numerous complexed forms that explain the substrate specificity as well as subtle kinetic differences between these two enzymes based on their crystal structures. We also re-examine the electrostatic influence of charged residues on the surface of the protein on the catalytic efficiency of the enzyme.     

The kinetic mechanism of human placental aldose reductase and aldehyde reductase II

The kinetic mechanism of NADPH-dependent aldehyde reductase II and aldose reductase, purified from human placenta, has been studied using L-glucuronate and DL-glyceraldehyde as their respective substrates. For aldehyde reductase II, the initial velocity and product inhibition studies (using NADP and gulonate) indicate that the enzyme reaction sequence is ordered with NADPH binding to the free enzyme and NADP being the last product to be released. Inhibition patterns using menadione (an analog of the aldehydic substrate) and ATP-ribose (an analog of NADPH) are also consistent with a compulsory ordered reaction sequence. Isotope effects of deuterium-substituted NADPH (NADPD) also corroborate the above reaction scheme and indicate that hydride transfer is not the sole rate-limiting step in the reaction sequence. For aldose reductase, initial velocity patterns, product, and dead-end inhibition studies indicate a random binding pattern of the substrates and an ordered release of product; the coenzyme is released last. A steady-state random mechanism is also consistent with deuterium isotope effects of NADPD on the reaction sequence catalyzed by this enzyme. However, the hydride transfer step seems to be more rate determining for aldose reductase than for aldehyde reductase II.