Investigations on the kinetic mechanism of octopine dehydrogenase. 1. Steady-state kinetics.

The kinetic mechanism of action of octopine dehydrogenase was investigated. This enzyme catalyses the reversible dehydrogenation of D-octopine to L-arginine and pyruvate, in the presence of nicotinamide-adenine dinucleotide. Initial velocity and product inhibition studies were carried out in both directions. Most of the results are consistent with a bi-ter sequential mechanism where NAD+ binds first to the enzyme followed by D-octopine, and the products are released in the order L-arginine, pyruvate and NADH. Various kinetic parameters were determined for each reactant at 33 degrees C, at pH 9.6 for NAD reduction, at pH 6.6 for NADH oxidation.     

Octopine dehydrogenase from Pecten maximus: steady-state mechanism

The steady-state kinetic mechanism of the reaction catalyzed by octopine dehydrogenase [N2-(1-carboxyethyl)-L-arginine:NAD+ oxidoreductase] was investigated at pH 6.9 and 9.2 by studies of substrate inhibition, analogue inhibition, and product inhibition. In the direction of octopine synthesis, the inhibition patterns in the presence of delta- guanidinovalerate and propionate show that NADH binds to the enzyme first followed by L-arginine and pyruvate which bind randomly. In the direction of octopine oxidation, the substrate patterns show that NAD binds to the enzyme before octopine in a rapid equilibrium fashion, and the product inhibition patterns show that the products L-arginine and pyruvate are released in a random fashion. Double, synergistic, substrate inhibition by L-arginine and pyruvate was shown to be due to binding (hypothetically of the imine) to the free enzyme and the enzyme-NAD complex. Furthermore, an alternate minor pathway was demonstrated which includes an enzyme-NADH-octopine complex and an enzyme-octopine complex.     

Temperature-determined enzymatic functions in octopine dehydrogenase.

We investigated the temperature dependence of several functions of octopine dehydrogenase, a monomeric enzyme extracted from the shell fish Pecten maximus L. We found that six enzymatic functions are temperature independent or change only negligibly with temperatue. These are the dissociation constants of three coenzyme complexes and the Michaelis Km values for NAD, NADH and one of the substrates (D-octopine). This is taken as an indication of a temperature-regulatory mechanism which enables the enzyme to maintain a constant level of NAD, NADH and D-octopine in binary and ternary complexes independent of fluctuations of the external temperature. This is discussed with reference to enzymes from other poikilotherms, which reportedly display similar biologically meaningful response to temperature. We also discuss the meaning of our data from a thermodynamic viewpoint. Considering that in a temperature-independent binding process only entropy changes contribute to the standard free-energy change, we speculate on possible molecular models which might account for our results. We also investigate the activation-energy parameters for the reaction catalyzed by octopine dehydrogenase, as obtained from the temperature dependence of V. It is found that octopine dehydrogenase, relative to other dehydrogenases, is provided with a rather low delta H not equal to, which enables the enzyme to change its turnover number by only a small factor in the temperature range 5--35 degrees C.     

Investigations on the kinetic mechanism of octopine dehydrogenase. 2. Location of the rate-limiting step for enzyme turnover.

The kinetic mechanism of octopine dehydrogenase has been investigated by stopped-flow and isotope replacement techniques. When the enzyme is saturated by substrate and coenzyme, both for NADH oxidation and NAD+ reduction, the stationary phase is preceded by a rapid burst. Under these saturation conditions, furthermore, the stationary phase shows a secondary isotope effect when 4S-[4(2)H]NADH is substituted for NADH and when (on the other reaction end) D-[2H] octopine is substituted for D-octopine. The data are taken to indicate that the rate-limiting step for enzyme turnover is a step following a very fast chemical transformation of the reagents. However, when the substrate concentration is lowered below the corresponding Km value keeping the coenzyme concentration at saturating levels, the time course of the reaction shows no burst and the stationary phase has a larger isotope effect. This indicated that under those non-saturating conditions, the enzyme turnover has a larger contribution than the hydrogen-transfer step. Changing the coenzyme concentration alone has very little or no effect on the amplitude of the burst or on the isotope effect. These features are discussed in terms of the other known kinetic properties of the enzyme, and in terms of analogous studies reported in the literature for other dehydrogenases.     

The kinetic locking-on strategy for bioaffinity purification: further studies with bovine liver glutamate dehydrogenase.

The locking-on strategy uses soluble analogues of the enzymes specific substrate to produce biospecific adsorption of individual NAD(P)(+)-dependent dehydrogenases on immobilized NAD(P)(+) derivatives, which is so selective that a single enzyme activity can be purified from crude cellular extracts in a single chromatographic step with yields approaching 100%. However, attempts to further develop and apply this strategy to the biospecific chromatographic purification of a range of NAD(P)(+)-dependent dehydrogenases revealed some anomalous chromatographic behavior and certain unexplained phenomenon. Much of this can be attributed to nonbiospecific interference effects. Identification and elimination of this interference is discussed in the present study focusing on bovine liver glutamate dehydrogenase (GDH; EC 1.4.1.3) as the "test" enzyme. Results further confirm the potential of the locking-on strategy for the rapid purification of NAD(P)(+)-dependent dehydrogenases and provide further insight into the parameters which should be considered during the development of a truly biospecific affinity chromatographic system based on the locking-on strategy. The kinetic mechanism of bovine liver GDH has been the topic of much controversy with some reports advocating a sequential ordered mechanism of substrate binding and others reporting a sequential random mechanism. Since the kinetic locking-on strategy is dependent on the target NAD(P)(+)-dependent dehydrogenase having an ordered sequential mechanism of substrate binding, the bioaffinity chromatographic behavior of bovine liver GDH using the locking-on tactic suggests that this enzyme has an ordered sequential mechanism of substrate binding under a variety of experimental conditions when NAD(+) is used as cofactor.     

Relationship between fluorescence and conformation of epsilonNAD+ bound to dehydrogenases.

This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle). The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated. It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme. Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form. Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching. In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity. The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+. For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate. One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites. The results and implications of this work are compared with those obtained recently by other authors.     

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.     

Allosterism and cooperativity in Pseudomonas aeruginosa GDP-mannose dehydrogenase

GDP-Mannose dehydrogenase catalyzes the formation of GDP-mannuronic acid, which is the monomeric unit from which the polysaccharide alginate is formed. Alginate is secreted by the pathogenic bacterium Pseudomonas aeruginosa and is believed to play an important role in the bacteria's resistance to antibiotics and the host immune response. We have characterized the kinetic behavior of GDP-mannose dehydrogenase in detail. The enzyme displays cooperative behavior with respect to NAD(+) binding, and phosphate and GMP act as allosteric effectors. Binding of the allosteric effectors causes the Hill coefficient for NAD(+) binding to decrease from 6 to 1, decreases K(1/2) for NAD(+) by a factor of 10, and decreases V(max) by a factor of 2. The cooperative binding of NAD(+) is also sensitive to pH; deprotonation of two residues with identical pK's of 8.0 is required for maximally cooperative behavior. The kinetic behavior of GDP-mannose dehydrogenase suggests that it must be at least hexameric under turnover conditions; however, dynamic light-scattering measurements do not provide a clear determination of the size of the active enzyme 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 studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Steady-state kinetic techniques have been used to investigate each of the reactions catalyzed by the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase, from Aerobacter aerogenes. The results of steady-state velocity studies in the absence of products, as well as product and dead-end inhibition studies, suggest that the prephenate dehydrogenase reaction conforms to a rapid equilibrium random mechanism which involes the formation of two dead-end complexes, viz, enzyme-NADH-prephenate and enzyme-NAD+-hydroxyphenylpyruvate. Chorismate functions as an activator of the dehydrogenase while both prephenate and hydroxyphenylpyruvate acted as competitive inhibitors in the mutase reaction. By contrast. bpth NAD+ and NADH function as activators of the mutase. Values of the kinetic parameters associated with the mutase and dehydrogenase reactions have been determined and the results discussed in terms of possible relationships between the catalytic sites for the two reactions. The data appear to be consistent with the enzyme having either a single site at which both reactions occur or two separate sites which possess similar kinetic properties.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

Bromopyruvate, a potential affinity label for octopine dehydrogenase

Bromopyruvate, an analogue of pyruvate, one of the substrates of octopine dehydrogenase, was tested as an inhibitor of the enzyme. Provided both the coenzyme and the second substrate, arginine, were present, bromopyruvate rapidly inactivated the enzyme. This inactivation was irreversible, obeyed pseudo-first order kinetics and exhibited a rate saturation effect. Pyruvate protected the enzyme against inactivation by bromopyruvate and these compounds competed for the same site. Bromopyruvate also behaved as a true substrate for the enzyme. This reagent thus exhibits the kinetic characteristics of a good affinity label for octopine dehydrogenase.     

Steady-state kinetics of coupled two-enzyme reactor with recycling of poly(ethylene glycol)-bound NAD

The kinetic properties of a continuous enzyme reactor containing rabbit muscle lactate dehydrogenase, horse liver alcohol dehydrogenase and poly(ethylene glycol)-bound NAD (PEG-NAD) were investigated experimentally and theoretically. The enzymes and PEG-NAD were retained in the reactor with an ultrafiltration membrane, and the substrates (pyruvate and ethanol) were fed continuously. The reactions of the dehydrogenases were coupled by the recycling of the cofactor. The steady-state concentration of L-lactate, one of the products, was measured under different experimental conditions and compared with the corresponding theoretical value. The theoretical value was calculated based on a simplified ordered bi-bi mechanism for the individual enzyme reactions, of which kinetic constants were determined by independent kinetic studies. Differences were found between the kinetic constants of the enzymes for NAD(H) and PEG-NAD(H). The steady-state values obtained by continuous operation were lower than those calculated, possibly due to the simplification made for the kinetic model; but there was general agreement between them in the dependence on the experimental conditions. The steady-state behavior of the enzyme reactor was explained semi-quantitatively by the simple kinetic model.     

Kinetic and spectroscopic studies of transhydrogenase activity and nucleotide site of lipoamide dehydrogenase

The kinetic behavior and spectroscopic characteristics of the nucleotide site(s) of lipoamide dehydrogenase have been investigated. Both subunits of the dimeric enzyme interact with NAD+. The binding of NAD+ is associated with a negative trough around 420-450 nm and a positive peak at 507 nm of the difference spectrum. The transhydrogenation between NADH and thionicotinamide nucleotide or acetylpyridine nucleotide is shown to proceed via a Ping Pong or an ordered Bi Bi mechanism, respectively, at pH above 7.0. Lowering pH or acetamidation lose the spectral characteristic of the positive peak of the enzyme-NAD+ complex with a concurrent change in the kinetic mechanism in the NADH+-acetylpyridine nucleotide transhydrogenation.     

Rhodococcus L-phenylalanine dehydrogenase: kinetics, mechanism, and structural basis for catalytic specificity

Phenylalanine dehydrogenase catalyzes the reversible, pyridine nucleotide-dependent oxidative deamination of L-phenylalanine to form phenylpyruvate and ammonia. We have characterized the steady-state kinetic behavior of the enzyme from Rhodococcus sp. M4 and determined the X-ray crystal structures of the recombinant enzyme in the complexes, E.NADH.L-phenylalanine and E.NAD(+). L-3-phenyllactate, to 1.25 and 1.4 A resolution, respectively. Initial velocity, product inhibition, and dead-end inhibition studies indicate the kinetic mechanism is ordered, with NAD(+) binding prior to phenylalanine and the products' being released in the order of ammonia, phenylpyruvate, and NADH. The enzyme shows no activity with NADPH or other 2'-phosphorylated pyridine nucleotides but has broad activity with NADH analogues. Our initial structural analyses of the E.NAD(+).phenylpyruvate and E.NAD(+). 3-phenylpropionate complexes established that Lys78 and Asp118 function as the catalytic residues in the active site [Vanhooke et al. (1999) Biochemistry 38, 2326-2339]. We have studied the ionization behavior of these residues in steady-state turnover and use these findings in conjunction with the structural data described both here and in our first report to modify our previously proposed mechanism for the enzymatic reaction. The structural characterizations also illuminate the mechanism of the redox specificity that precludes alpha-amino acid dehydrogenases from functioning as alpha-hydroxy acid dehydrogenases.     

Depolymerisation of F-actin to G-actin and its repolymerisation in the presence of analogs of adenosine triphosphate.

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring. The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect. Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide--8-bromoadenine dinucleotide, nicotinamide--8-thioadenine dinucleotide and nicotinamide 1: N6-ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes. Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding. The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary by not ternary complexes. The case of 1,4,5,6-tetrahydronicotinamide--adenine dinucleotide which does not give ternary complexes like NADH is discussed. The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described. The protective effects of coenzyme analogues towards essential -SH group were also studied.     

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.     

Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1.

A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.     

Mechanism of action of Drosophila melanogaster alcohol dehydrogenase.

Reported kinetic pH dependence data for alcohol dehydrogenase from Drosophila melanogaster are analyzed with regard to differences in rate behaviour between this non-metallo enzyme and the zinc-containing liver alcohol dehydrogenase present in vertebrates. For the Drosophila enzyme a mechanism of action is proposed according to which catalytic proton release to solution during alcohol oxidation occurs at the binary-complex level as an obligatory step preceding substrate binding. Such proton release involves an ionizing group with a pKa of about 7.6 in the enzyme.NAD+ complex, tentatively identified as a tyrosyl residue. The ionized form of this group is proposed to participate in the binding of alcohol substrates and to act as a nucleophilic catalyst of the subsequent step of hydride ion transfer from the bound alcohol to NAD+. Herein lie fundamental mechanistic differences between the metallo and non-metallo short chain alcohol dehydrogenases.     

Bovine lens aldehyde dehydrogenase. Kinetics and mechanism.

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.