Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

• 1.1. A NAD⁺-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
• 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD⁺-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na⁺ than with K⁺ in the amination reaction while the reverse is true in the deamination reaction.
• 3.3. The apparent K_m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD⁺, 0.30 mM.
• 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.

     

Glutamate dehydrogenase of lupin nodules: Purification and properties

Glutamate dehydrogenase, GDH (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD⁺ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD⁺. When NAD⁺ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD⁺.     

Novel halophilic 2-aminobutyrate dehydrogenase from Halobacterium saccahrovorum DSM 1137

A halophilic NAD⁺-dependent 2-aminobutyrate dehydrogenase (EC1.4.1.1) was purified to homogeneity from a crude extract of an extreme halophile, Halobacterium saccharovorum DSM 1137, with a 30% yield. The enzyme had a molecular mass of about 160 kDa and consisted of four identical subunits. It retained more than 70% of the activity after heating at 60 °C for 1 h and kept it at 30 °C for 8 months in the presence of 2 M NaCl. The enzyme showed maximum activity in the presence of 2 M RbCl or KCl. The enzyme required NAD⁺ as a coenzyme and used -2-aminobutyrate, -alanine, and -norvaline as substrates. The best substrate was -2-aminobutyrate. The optimum pH was 9.3 for the oxidative deamination of -2-aminobutyrate and 8.6 for the reductive amination of 2-ketobutyrate. The Michaelis constants were 1.2 mM for -2-aminobutyrate, 0.16 mM for NAD⁺, 0.012 mM for NADH, 0.78 mM for 2-ketobutyrate, and 500 mM for ammonia in the presence of 2 M KCl. The K_m values for the substrates depended on the concentration of KCl, and the K_m values decreased under high salt conditions.     

Alanine dehydrogenase and its applications – A review

Alanine dehydrogenase (AlaDH) (E.C.1.4.1.1) is a microbial enzyme that catalyzes a reversible conversion of L-alanine to pyruvate. Inter-conversion of alanine and pyruvate by AlaDH is central to metabolism in microorganisms. Its oxidative deamination reaction produces pyruvate which plays a pivotal role in the generation of energy through the tricarboxylic acid cycle for sporulation in the microorganisms. Its reductive amination reaction provides a route for the incorporation of ammonia and produces L-alanine which is required for synthesis of the peptidoglycan layer, proteins, and other amino acids. Also, AlaDH helps in redox balancing as its deamination/amination reaction is linked to the reduction/oxidation of NAD⁺/NADH in microorganisms. AlaDH from a few microorganisms can also reduce glyoxylate into glycine (aminoacetate) in a nonreversible reaction. Both its oxidative and reductive reactions exhibit remarkable applications in the pharmaceutical, environmental, and food industries. The literature addressing the characteristics and applications of AlaDH from a wide range of microorganisms is summarized in the current review.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

The effect of anions on the reaction catalyzed by lupine-nodule glutamate dehydrogenase

The kinetics of the reductive amination reaction of lupine-nodule glutamate dehydrogenase (l-glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2) were found to vary with the identity of the ammonium salt which was used as a substrate. Normal Michaelis-Menten kinetics were obtained with (NH₄)₂SO₄ but when NH₄Cl or NH₄-acetate was varied apparent substrate inhibition was observed. Linear double-reciprocal plots were obtained with NH₄Cl and NH₄-acetate, however, if the concentration of Cl^? or acetate was maintained constant by adding KCl or K-acetate. Chloride and acetate were subsequently found to cause linear noncompetitive inhibition with respect to NH₄⁺ and the apparent substrate inhibition by NH₄Cl and NH₄-acetate can be explained as the result varying a substrate and a noncompetitive inhibitor in constant ratio. Other anions were also found to be inhibitors of the glutamate dehydrogenase reaction; I^? caused parabolic noncompetitive inhibition with respect to NH₄⁺ and NO₃^? caused slope-parabolic noncompetitive inhibition with respect to all three substrates of the reductive amination reaction. For the oxidation deamination reaction, Cl^? was a linear competitive inhibitor with respect to both NAD and l-glutamate whereas NO₃^? caused parabolic competitive inhibition with respect to these reactants. To explain the results, it is proposed that anions bind to an allosteric site and cause a change in some of the rate constants of the reaction. Specifically, the results are consistent with anions causing decreases in the rates of association of NADH and 2-oxoglutarate with the enzyme and an increase in the rate of dissociation of NAD.     

Glutamate dehydrogenase of lupin nodules: Kinetics of the deamination reaction

The reaction of glutamate dehydrogenase (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) from lupin nodules has been investigated in the direction of deamination by means of steady state velocity studies in the absence of products and inhibition studies with products and substrate analogs. The results are qualitatively and quantitatively consistent with a fully ordered reaction mechanism in which NAD⁺ binds to the enzyme first followed by l-glutamate. The order of product release is proposed to be NH₄⁺ followed by 2-oxoglutarate and then NADH. In addition, product inhibition data indicate the formation of an enzyme-NAD-oxoglutarate dead-end complex.     

l-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea

The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. l-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP⁺-dependent enzymes from chloroplasts and was separated from the NAD⁺-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis–Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K _m for oxaloacetate was 20 μM; the K _m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD⁺ increased with pH but there was very little activity with NADP⁺. At pH 7.0, the K _m for l-malate was 5 mM and the K _m for NAD⁺ was 24 μM. The reductive activity was quite insensitive to inhibition by l-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10–20 times higher than the oxidative activity. These results indicate that the l-malate dehydrogenase in N. europaea is similar to other NAD⁺-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Characterization of an NAD(P)+-dependent meso-diaminopimelate dehydrogenase from Thermosyntropha lipolytica

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP⁺-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP⁺ and NAD⁺ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5–9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.     

Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase

The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD⁺ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD⁺. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD⁺ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase_TOL with dioxygen and thus present a solution toward conflicting requirements.     

Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18.

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.     

Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus

Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the K_m values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Interaction of the enzyme with coenzymes and coenzyme analogs.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD⁺ or NADP⁺ as coenzyme. Kinetic studies showed that NAD⁺ and NADP⁺ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP⁺ and 50% for NAD⁺. The dissociation constant for NADP⁺, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined K_m of 5.7 μm and K_i of 5 μm. The dissociation constant for NAD⁺ is 2.5 mm, which is 24 times larger than the previously determined K_m of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD⁺ but not for NADP⁺. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD⁺ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD⁺; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined K_i is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP⁺-competitive and NAD⁺-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD⁺ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.