The simultaneous determination of NAD(H) and NADP(H) utilization by glutamate dehydrogenase

Glutamate dehydrogenase (GDH) from vertebrates is unusual among NAD(P)H-dependent dehydrogenases in that it can use either NAD(H) or NADP(H) as cofactor. In this study, we measure the rate of cofactor utilization by bovine GDH when both cofactors are present. Methods for both reaction directions were developed, and for the first time, to our knowledge, the GDH activity has been simultaneously studied in the presence of both NAD(H) and NADP(H). Our data indicate that NADP(H) has inhibitory effects on the rate of NAD(H) utilization by GDH, a characteristic of GDH not previously recognized. The response of GDH to allosteric activators in the presence of NAD(H) and NADP(H) suggests that ADP and leucine moderate much of the inhibitory effect of NADP(H) on the utilization of NAD(H). These results illustrate that simple assumptions of cofactor preference by mammalian GDH are incomplete without an appreciation of allosteric effects when both cofactors are simultaneously present.     

克雷伯杆菌甘油脱氢酶和1,3-丙二醇氧化还原酶的动力学机制

本研究主要对克雷伯杆菌甘油转化1,3-丙二醇代谢途径中的2个关键酶甘油脱氢酶(GDH)、1,3-丙二醇氧化还原酶(PDOR)反应机制和动力学进行了研究。首先,通过初速度和产物抑制动力学研究确定了GDH、PDOR双底物酶促反应机制为有序BiBi机制,明确了由反应物消耗到产物生成之间的历程。其次,建立了GDH、PDOR双底物酶促反应动力学模型,由动力学模型可知,在偶合反应中,如果GDH和PDOR酶量相同,GDH氧化反应成为限速反应,而辅酶I将主要以氧化型NAD+形式存在。动力学信息为酶法合成1,3-丙二醇和代谢工程研究提供理论指导。     

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.     

3Alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni: kinetic properties with NAD and its thionicotinamide analogue.

The kinetics of 3alpha-hydroxysteroid : NAD oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) have been investigated. The kinetic analysis based on initial activity measurements and product inhibition studies, indicates that the addition of substrate to the enzyme and the release of products from it, follows an obligatory order (ordered bi bi mechanism). The ability of the enzyme to utilize the thionicotinamide analogue of NAD (sNAD) as cofactor has been investigated using various 3alpha-hydroxysteroids from both the C19, C21, and C24 series. The results show that the reaction velocity with sNAD as the cofactor is generally lower than with NAD. The decrease, however, varies considerably, being negligible with some steroids such as litocholic acid and deoxycholic acid and very pronounced with other such as tetrahydrocortisol and tetrahydrocortisone. The introduction of an 11beta-hydroxy or an 11-oxo group into the steroid molecule significantly reduces the ability of the enzyme to attack the 3alpha-hydroxy group. No such effect could be seen when the 11-hydroxy group was in the alpha-position. The results also indicate that, whereas NAD can serve as cofactor for both the monomeric and the dimeric forms of the enzyme, sNAD only acts as cofactor for the monomeric form. Thus sNAD is a valuable tool for the study of the reversible, concentration-depenedent monomeric-dimeric transition of the 3alpha-hydroxysteroid dehydrogenase.     

Effect of thermal acclimation on subunit cooperativity in Palaemon serratus glutamate dehydrogenase

Glutamate dehydrogenase (GDH) from the abdominal muscle of the shrimp Palaemon serratus displays a complex cooperatively pattern with respect to the cofactor (NAD) and substrate (glutamate) concentrations: at low concentrations negative cooperativitiy is predominant whereas positive cooperativity prevails at high concentrations. GDH is sensitive to thermal variations of the environment and to conditions of thermal acclimation. The maximum cooperativity indexes (positive and negative) are obtained at 13° for NAD irrespective of the acclimation temperature (13 or 18°). In contrast, for glutamate, positive cooperativity is only observed at temperatures near the acclimation temperature. At 13° for animals adapted at 18° and at 27° for animals adapted at 13° a complete loss of subunit cooperativity and a Michaelian kinetic pattern are observed.     

A "stripping" ligand tactic for use with the kinetic locking-on strategy: its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases.

The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27).     

Properties of partially purified saccharopine dehydrogenase from human placenta.

Saccharopine dehydrogenase was previously purified 380-fold from human placenta. The enzyme was shown to catalyze the formation of α-aminoadipic-δ-semialdehyde and glutamate from saccharopine, to have a molecular weight of 480,000 on gel filtration, and not to be separable from l-lysine-α-ketoglutarate reductase. Additional properties of the saccharopine dehydrogenase are now described. The pH optimum for the conversion of saccharopine to glutamate and α-aminoadipic-δ-semialdehyde is 8.5 in Tris-HCl buffer and 8.9 in 2-amino-2-methyl-1,3-propanediol buffer. The specificity of the enzyme for Saccharopine and NAD and the inhibition by glutamate and product analogs were tested. It was found the NADP was the only cofactor that could replace NAD in the enzyme reaction and that several NAD analogs were reaction inhibitors. Glutamate was found to be only moderately effective as an inhibitor. Initial velocity studies revealed that the enzyme has an ordered reaction mechanism. The true K_m values for saccharopine and NAD are 1.15 mm and 0.0645 mm, respectively.     

Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol(-1), mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.     

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.     

Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate,cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.     

The cofactor preference of glucose-6-phosphate dehydrogenase from Escherichia coli--modeling the physiological production of reduced cofactors

In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol(-1). In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the ω-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rR(NAD)) and NADP/NADPH (rR(NADP)). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR(NADP), but NADH production remains low within the range feasible for rR(NAD). Nevertheless, we show that certain combinations of rR(NADP) and rR(NAD) sustain greater levels of NADH production over NADPH.     

Self-assembled CNTs/CdS/dehydrogenase hybrid-based amperometric biosensor triggered by photovoltaic effect

A novel multi-components hybrid material, self-assembled quantum dots (CdS) and glutamate dehydrogenase (GDH) onto multiwall carbon nanotubes (CNTs), was designed for amperometric biosensing system. The zeta-potential and transmission electron microscopy (TEM) analyses confirmed the uniform growth of the CdS/GDH onto carboxyl-functionalized CNTs. Compared with the single CdS, the resulting hybrid material showed more efficient generation of photocurrent upon illumination. The incident light excites CdS and generates charge carriers, and then CNTs facilitates the charge transfer. For dehydrogenase-based biosensor, normally, the cofactor of beta-nicotinamide adenine dinucleotide (NAD(+)) or beta-nicotinamide adenine dinucleotide phosphate (NADP(+)) is necessary. Furthermore, we found the photovoltaic effect of CNTs/CdS/GDH can trigger the dehydrogenase enzymatic reaction in the absence of the NAD(+) or NADP(+) cofactors. The electrochemical experiment results also demonstrate that the cofactor-independent dehydrogenase biosensing system had series attractive characteristics, such as a good sensitivity (11.9nA/muM), lower detection limit (up to 50nM), an acceptable reproducibility and stability. These studies aid in understanding the combination of the semiconductor nanohybrids (CNTs/QDs, etc.) and biomolecules (enzymes, etc.), which has potential for the applications in biosensor, biofuel cell, biomedical and other bioelectronics field.     

Characterization of Peptostreptococcus asaccharolyticus glutamate dehydrogenase purified by dye-ligand chromatography

Glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase (deaminating); EC 1.4.1.2) has been purified from Peptostreptococcus asaccharolyticus in a single step using dye-ligand chromatography. The enzyme (GDH) was present in high yields and was stabilized in crude extracts. A subunit molecular weight of 49000 +/- 500 was determined by SDS polyacrylamide gel electrophoresis and six bands were obtained after cross-linking the subunits with dimethyl suberimidate. This bacterial GDH was predominantly NAD+-linked, but was able to utilize both NADP+ and NADPH at 4% of the rates with NAD+ and NADH, respectively. An investigation of the amino acid specificity revealed some similarities with GDH from mammalian sources and some clear differences. The values of apparent Km for the substrates ammonia, 2-oxoglutarate, NADH, NAD+ and glutamate were 18.4, 0.82, 0.066, 0.031 and 6 mM, respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength.     

Crystal structure of uronate dehydrogenase from Agrobacterium tumefaciens

Uronate dehydrogenase from Agrobacterium tumefaciens (AtUdh) belongs to the short-chain dehydrogenase/reductase superfamily and catalyzes the oxidation of D-galacturonic acid and D-glucuronic acid with NAD(+) as a cofactor. We have determined the crystal structures of an apo-form of AtUdh, a ternary form in complex with NADH and product (substrate-soaked structure), and an inactive Y136A mutant in complex with NAD(+). The crystal structures suggest AtUdh to be a homohexamer, which has also been observed to be the major form in solution. The monomer contains a Rossmann fold, essential for nucleotide binding and a common feature of the short-chain dehydrogenase/reductase family enzymes. The ternary complex structure reveals a product, D-galactaro-1,5-lactone, which is bound above the nicotinamide ring. This product rearranges in solution to D-galactaro-1,4-lactone as verified by mass spectrometry analysis, which agrees with our previous NMR study. The crystal structure of the mutant with the catalytic residue Tyr-136 substituted with alanine shows changes in the position of Ile-74 and Ser-75. This probably altered the binding of the nicotinamide end of NAD(+), which was not visible in the electron density map. The structures presented provide novel insights into cofactor and substrate binding and the reaction mechanism of AtUdh. This information can be applied to the design of efficient microbial conversion of D-galacturonic acid-based waste materials.     

Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies

Five different immobilized NAD+ derivatives were employed to compare the behavior of four amino acid dehydrogenases chromatographed using kinetic-based enzyme capture strategies (KBECS): S6-, N6-, N1-, 8'-azo-, and pyrophosphate-linked immobilized NAD+. The amino acid dehydrogenases were NAD+-dependent phenylalanine (EC 1.4.1.20), alanine (EC 1.4.1.1), and leucine (EC 1.4.1.9) dehydrogenases from various microbial species and NAD(P)+-dependent glutamate dehydrogenase from bovine liver (GDH; EC 1.4.1.3). KBECS for bovine heart L-lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1) were also applied to assist in a preliminary assessment of the immobilized cofactor derivatives. Results confirm that the majority of the enzymes studied retained affinity for NAD+ immobilized through an N6 linkage, as opposed to an N1 linkage, replacement of the nitrogen with sulfur to produce an S6 linkage, or attachment of the cofactor through the C8 position or the pyrophosphate group of the cofactor. The one exception to this was the dual-cofactor-specific GDH from bovine liver, which showed no affinity for N6-linked NAD+ but was biospecifically adsorbed to S6-linked NAD+ derivatives in the presence of its soluble KBEC ligand. The molecular basis for this is discussed together with the implications for future development and application of KBECS.     

Continuous regeneration of NAD(P)+ by flavins covalently bound to sepharose.

Various flavins, FMN, FAD, and acriflavin, were immobilized to Sepharose using several different coupling methods. The only product stable enough to permit extended studies was acriflavin coupled to epoxy-substituted Sepharose. The nonenzymic oxidizing capacity towards NAD(P) H was investigated and a 25% specific activity, compared to that of free acriflavin, was observed. The reduced acriflavin was immediately auto-reoxidized in air and could thus be reused. It was shown that acriflavin-Sepharose preparations function as NAD(P)H oxidizing agents in a number of different dehydrogenase systems including lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), alanine dehydrogenase (alaDH), and glutamate dehydrogenase (GDH). The amount of expensive coenzyme necessary for high product formation of such systems was thereby markedly reduced.     

Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+

This study reevaluates the potential for direct coupling of NAD(P)(+) to a carboxylate-terminating spacer arm using carbodiimide-promoted coupling in an attempt to develop a greatly simplified synthetic method for cofactor immobilization that would support the more widespread adoption of kinetic-based enzyme capture (KBEC) strategies for protein purification applications and protein-detecting arrays/proteomic studies. Direct coupling of NAD(+) to epoxy (1,4-butanediol diglycidyl ether)-activated Sepharose is also described. Depending on the synthetic method used, the position of attachment of cofactor is concluded to be primarily through the pyrophosphate or ribosyl hydroxyl groups. Total substitution levels varied from 0.5 to 2 micromol/g wet weight with 28-67% accessibility. Model bioaffinity chromatographic studies employing KBEC strategies are reported for bovine heart L-lactate dehydrogenase, yeast alcohol dehydrogenase, l-phenylalanine dehydrogenase from Sporosarcina, glutamate dehydrogenase (GDH) from Candida utilis, and GDH from bovine liver. The NAD(+) derivative prepared using epoxy-activated Sepharose shows most potential for further development based on total substitution levels, the apparent absence of nonbiospecific interference, reversible biospecific adsorption of some of the test enzymes using soluble KBEC/stripping ligand tactics, and the relative simplicity of the synthetic method.     

Continuous production of L-tert-leucine in series of two enzyme membrane reactors

The L-tert-leucine synthesis was performed continuously in series of two enzyme-membrane reactors by reductive amination of trimethylpyruvate with leucine dehydrogenase. The necessary “native” cofactor NADH is regenerated with the aid of a second enzyme, formate dehydrogenase. Considering detailed kinetic studies of initial reaction rates under conditions relevant to the process a kinetic model was developed. The model shows that the overall reaction rate is strongly inhibited by the reaction product. The reactor's models combine the mass balances and proposed kinetic equations. The model adequacy was verified by using it to simulate the experiments and by comparing experimental and computed conversion, space-time yield and enzyme consumption. The calculations for the three reactor's types (batch, single CSTR and a cascade of two CSTRs in series) were compared. The results showed that a single CSTR is no favourable reactor configuration due to the very strong product inhibition. Space-time yield drops from 560 g litre^?1 day^?1 in a batch reactor to 110 g litre^?1 day^?1 in a single CSTR at the highest conversion of 98%. At the conversion of 95% the difference in biocatalyst costs between batch and two CSTR in series is negligible. Therefore the use of two enzyme membrane reactors in series was proposed. The modelling in this work shows that the optimisation of the quantity of the enzyme used results in a minimisation of the biocatalyst costs.     

The activities of the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells are kinetically independent

The bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells has very different kinetic properties from the larger NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase present in all mammalian cells. The NAD-dependent dehydrogenase is unique in that it requires formation of a magnesium.enzyme complex to allow addition of the first substrate, NAD+. It catalyzes an equilibrium ordered kinetic mechanism that has methylenetetrahydrofolate as the last reactant to add and NADH as the last product released. The NADP-dependent dehydrogenase has the same order of addition of substrates, but NADPH is released prior to methenyltetrahydrofolate. The dehydrogenase-cyclohydrolase activities of both enzymes channel methenyltetrahydropteroylglutamate intermediates with the same efficiency which is unaffected by the number of glutamyl residues in the methylenetetrahydrofolate substrate. However, the cyclohydrolase activity of the bifunctional protein is kinetically independent of its dehydrogenase activity, as supported by its lack of inhibition by NAD+, whereas NADP+ strongly inhibits that of the NADP-dependent enzyme. This difference is further demonstrated by the observation that conversion of formyltetrahydrofolate to methylenetetrahydrofolate in the presence of reduced pyridine nucleotide is catalyzed readily only by the bifunctional enzyme.     

Enabling multienzyme biocatalysis using nanoporous materials

Multistep reactions catalyzed by a covalently immobilized enzyme-cofactor-enzyme system were achieved. Lactate dehydrogenase (LDH), glucose dehydrogenase (GDH), and cofactor NADH were incorporated into two porous silica glass supports. One of the glass supports had pores of 30 nm in diameter, while the other was of 100-nm pore size. Effective shuttling of the covalently bound NADH between LDH and GDH was achieved, such that regeneration cycles of NADH/NAD(+) were observed. The glass of 30-nm pore size afforded enzyme activities that were about twice those observed for the glass of 100-nm pore size, indicating the former provided better enzyme-cofactor integration. The effect of the size of spacers was also examined. The use of longer spacers increased the reaction rates by approximately 18 times as compared to those achieved with glutaraldehyde linkage. It appeared that the concave configuration of the nanopores played an important role in enabling the multistep reactions. The same multienzyme system immobilized on nonporous polystyrene particles of 500-nm diameter was only approximately 2% active as the glass-supported system. It is believed that the nanoporous structure of the glass supports enhances the molecular interactions among the immobilized enzymes and cofactor, thus improving the catalytic efficiency of the system.