A new assay for L-aspartate:2-oxoglutarate aminotransferase.

A new enzymatic assay for aspartate aminotransferase is presented. The 2-oxoglutarate formed in transamination between l-glutamate and oxalacetate was determined in a system coupled with hydroxyglutarate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. The method allowed accurate determination of the initial velocity of the reaction, which was proportional to the enzyme concentration. The Michaelis constants of pig heart cytosolic aspartate aminotransferase for l-glutamate and oxalacetate and the amino acceptor specificity using l-glutamate as an amino donor were determined. The method was applicable to the determination of the enzyme activity in various materials including rat serum and bacterial crude extract.     

Enzymic methods for the micro assay of D-mannose,D-glucose,D-galactose,and L-fucose from acid hydrolyzates of glycoproteins

Enzymic methods of micro assay have been described for four neutral sugars commonly present in glycoproteins and glycolipids. These assays can be applied to glycoprotein hydrolyzates without prior purification of individual sugars.d-Mannose is assayed by first phosphorylating the sugar in the presence of hexokinase and then measuring the formation of ADP by the use of pyruvate kinase and lactic dehydrogenase. This assay is not specific for d-mannose since both d-glucose and d-glucosamine can be phosphorylated by hexokinase. It is possible to remove d-glucosamine prior to hexokinase treatment by passage through a Dowex 50-X8 (H⁺) column. The effect of d-glucose in the sample can be corrected for by measuring d-glucose with d-glucose-6-phosphate dehydrogenase, an assay which is highly specific for d-glucose.d-Galactose and l-fucose are measured by their respective dehydrogenases. Neither of these dehydrogenases is affected by sugars commonly found in glycoproteins or glycolipids, nor by the presence of a partial acid hydrolyzate of bovine serum albumin. The methods described enable detection of 0.025 μmole of d-mannose, d-glucose, d-galactose, or l-fucose in a glycoprotein digest. The methods can theoretically be made even more sensitive by the use of fluorometric techniques.     

Investigations on the structural perturbations induced by the binding of mercuric chloride to L-glutamate dehydrogenase.

Three mercuric chloride binding sites are identified on l-glutamate dehydrogenase. In the presence of EDTA, the binding of two mercuric chloride molecules per subunit induces the dissociation of the polyhexamers into hexamers. The physical and catalytic properties of this modified hexamer are similar to those of the native enzyme. This induced dissociation of the enzyme is probably the result of an exclusive binding of the mercurial to the free hexamer, and the dissociation velocity does not appear to be rate limited by the binding reaction of the mercurials. The third mercuric chloride binding site is protected by both EDTA and l-glutamate. The binding of HgCl₂ to this site leads to the complete inactivation of the protein. There is no overlap between these modifications of l-glutamate dehydrogenase and the two previously described modifications of the enzyme by mercurial.     

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of ¹⁴CO₂ from l-[1-¹⁴C]fucose and d-[1-¹⁴C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-¹⁴C]fucose or of d-[1-¹⁴C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more ¹⁴CO₂ from l-[1-¹⁴C]fucose or d-[1-¹⁴C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies.     

The physical properties of NAD-dependent l-fucose dehydrogenase from sheep liver

Sheep liver l-fucose (d-arabinose) dehydrogenase has been purified to homogeneity as indicated by polyacrylamide disc-gel electrophoresis and sedimentation velocity ultracentrifugation experiments. The enzyme possesses an apparent molecular weight of 123,000 and is composed of four subunits of molecular weight approximately 30,000. The pI of the enzyme is 5.8. The enzyme is stable at high temperatures, retaining 65% of its original activity after 15 min at 60 °C. High ionic strength (μ = 1.0–1.3) in the assay medium stimulates the enzymatic activity and lowers the pH values at which maximal enzymatic activity is observed.     

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.     

Bacterial cell-surface displaying of thermo-tolerant glutamate dehydrogenase and its application in l-glutamate assay

In this paper, glutamate dehydrogenase (Gldh) is reported to efficiently display on Escherichia coli cell surface by using N-terminal region of ice the nucleation protein as an anchoring motif. The presence of Gldh was confirmed by SDS-PAGE and enzyme activity assay. Gldh was detected mainly in the outer membrane fraction, suggesting that the Gldh was displayed on the bacterial cell surface. The optimal temperature and pH for the bacteria cell-surface displayed Gldh (bacteria-Gldh) were 70 °C and 9.0, respectively. Additionally, the fusion protein retained almost 100% of its initial enzymatic activity after 1 month incubation at 4 °C. Transition metal ions could inhibit the enzyme activity to different extents, while common anions had little adverse effect on enzyme activity. Importantly, the displayed Gldh is most specific to l-glutamate reported so far. The bacterial Gldh was enabled to catalyze oxidization of l-glutamate with NADP⁺ as cofactor, and the resultant NADPH can be detected spectrometrically at 340 nm. The bacterial-Gldh based l-glutamate assay was established, where the absorbance at 340 nm increased linearly with the increasing l-glutamate concentration within the range of 10 − 400 μM. Further, the proposed approach was successfully applied to measure l-glutamate in real samples.     

An assay for free l-fucose in the presence of the bound sugar

An assay for free l-fucose in the presence of the bound form is described. It is based upon the enzymatic conversion of free l-fucose to l-fuculose which is measured by the cysteine-carbazole reaction.The assay is sensitive, relatively specific, and is well suited for the measurement of fucosidases.     

A high-throughput assay for screening l- or d-amino acid specific aminotransferase mutant libraries

Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the l-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either l- or d-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 d-amino acid aminotransferase displaying a decrease in k_cat/K_M of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate l-leucine than l-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering.     

Structural basis of substrate selectivity of Δ-pyrroline-5-carboxylate dehydrogenase (ALDH4A1): Semialdehyde chain length

The enzyme Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase (aka P5CDH and ALDH4A1) is an aldehyde dehydrogenase that catalyzes the oxidation of γ-glutamate semialdehyde to l-glutamate. The crystal structures of mouse P5CDH complexed with glutarate, succinate, malonate, glyoxylate, and acetate are reported. The structures are used to build a structure-activity relationship that describes the semialdehyde carbon chain length and the position of the aldehyde group in relation to the cysteine nucleophile and oxyanion hole. Efficient 4- and 5-carbon substrates share the common feature of being long enough to span the distance between the anchor loop at the bottom of the active site and the oxyanion hole at the top of the active site. The inactive 2- and 3-carbon semialdehydes bind the anchor loop but are too short to reach the oxyanion hole. Inhibition of P5CDH by glyoxylate, malonate, succinate, glutarate, and l-glutamate is also examined. The K_i values are 0.27 mM for glyoxylate, 58 mM for succinate, 30 mM for glutarate, and 12 mM for l-glutamate. Curiously, malonate is not an inhibitor. The trends in K_i likely reflect a trade-off between the penalty for desolvating the carboxylates of the free inhibitor and the number of compensating hydrogen bonds formed in the enzyme-inhibitor complex.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.     

Thermophilic l-fucose isomerase from Thermanaeromonas toyohensis for l-fucose synthesis from l-fuculose

The beneficial biological properties of l-fucose have extended its commercial application potential in pharmaceutical, cosmetic, and food industries. Enzymatic production of l-fucose with l-fucose isomerase (l-FucI) is considered a selective, green, and efficient strategy. Efficient sugar production requires thermophilic enzymes with increased reaction rate, reduced risk of microbial contamination, and high sugar solubility. No study has evaluated the applicability of thermophilic l-FucI for l-fucose production. In this study, we explored the biochemical properties of a thermostable l-FucI from Thermanaeromonas toyohensis (TtFucI) using l-fuculose as a substrate. The recombinant TtFucI exhibited thermophilicity and optimum activity at 70 °C. The specific activity, K_m, and k_cat toward l-fuculose were 199.8 U/mg, 33.4 mM, and 901.7 s⁻¹, respectively. Mn²⁺ ions increased the activity of the enzyme by ∼10 times and enhanced its thermal stability. Our study, on l-fucose synthesis by thermostable l-FucI, suggests the potential application of this enzyme for the industrial production of l-fucose.     

A simple enzymatic method for the analysis of macromolecular fucose in biological materials

A sensitive enzymatic method employing l-fucose dehydrogenase has been used for the measurement of the amounts of fucose in 100–500 μg of plasma membrane protein and 10–100 μg of porcine submaxillary mucin. The assay showed linearity between 0 and 20 nmol of α-l-fucose when measuring NADH fluorescence. The fucose values obtained for the plasma membrane and submaxillary mucin correspond well with those obtained by gas-liquid chromatography.     

Synthesis and evaluation of novel photoreactive α-amino acid analog carrying acidic and cleavable functions

A novel photoreactive α-amino acid bearing an acidic residue and a cleavable diazirine was developed. To mimic common acidic α-amino acids, the residue was designed to be N-acylsulfonamide that possesses an acidic proton and is able to dissociate under the physiological conditions. The inhibition assay of its biotin-tagged derivative with glutamyl endopeptidase from Staphylococcus aureus V8 strain revealed a Ki_app value of 162 μM, which is slightly higher than the K_m value of a common substrate. Upon UV irradiation, this derivative specifically photolabeled glutamyl endopeptidase, l-glutamate dehydrogenase, glutamic oxalacetic transaminase, and l-glutamine synthetase, all the enzymes exhibit high affinity toward acidic α-amino acids. In addition, N-acylsulfonamide group functioned as a cleavable linker in mild basic solution after a brief N-alkylation. Either the multifunctional nature or the simple structure of this acidic α-amino acid surrogate would be useful as versatile photoreactive building block.     

Purification, properties, and regulation of glutamic dehydrogenase of Bacillus licheniformis

Cell-free extracts of Bacillus licheniformis and B. cereus were found to contain high specific activities of nicotinamide adenine dinucleotide phosphate (NADP)-dependent-l-glutamate dehydrogenase [EC 1.4.1.4; l-glutamate: NADP oxidoreductase (deaminating)]. Maximum specific activities were found in extracts of cells during the late exponential phase of growth when ammonium ion served as the sole source of nitrogen. Extremely low specific activities were detected throughout the growth cycle when l-glutamate or Casamino Acids served as the source of carbon and nitrogen. The enzyme was purified 55-fold from crude extracts of B. licheniformis, and apparent kinetic constants were determined. Sigmoidal saturation kinetics were not observed, and various adenylates had no effect on the enzyme. Repression of enzyme synthesis during growth on l-glutamate or Casamino Acids was partially overcome by additions of glucose or pyruvate, and this apparent derepression was totally abolished by inhibitors of ribonucleic acid and protein synthesis. Similarly, additions of l-glutamate or Casamino Acids to cells growing on glucose-ammonium ion resulted in strong repression of enzyme synthesis. It is suggested that the enzyme serves an anabolic role in metabolism. Nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase activity was not detected in five species of Bacillus, irrespective of nutritional conditions or of the physiological age of cells.     

Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

Comparative Studies of Enzymes Related to Serine Metabolism in Higher Plants

THE FOLLOWING ENZYMES RELATED TO SERINE METABOLISM IN HIGHER PLANTS HAVE BEEN INVESTIGATED: 1) d-3-phosphoglycerate dehydrogenase, 2) phosphohydroxypyruvate:l-glutamate transaminase, 3) d-glycerate dehydrogenase, and 4) hydroxypyruvate:l-alanine transaminase. Comparative studies on the distribution of the 2 dehydrogenases in seeds and leaves from various plants revealed that d-3-phosphoglycerate dehydrogenase is widely distributed in seeds in contrast to d-glycerate dehydrogenase, which is either absent or present at low levels, and that the reverse pattern is observed in green leaves.The levels of activity of the 4 enzymes listed above were followed in different tissues of the developing pea (Pisum sativum, var. Alaska). In the leaf, from the tenth to seventeenth day of germination, the specific activity of d-glycerate dehydrogenase increased markedly and was much higher than d-3-phosphoglycerate dehydrogenase which remained relatively constant during this time period. Etiolation resulted in a decrease in d-glycerate dehydrogenase and an increase in d-3-phosphoglycerate dehydrogenase activities. In apical meristem, on the other hand, the level of d-3-phosphoglycerate dehydrogenase exceeded that of d-glycerate dehydrogenase at all time periods studied. Low and decreasing levels of both dehydrogenases were found in epicotyl and cotyledon. The specific activities of the 2 transaminases remained relatively constant during development in both leaf and apical meristem. In general, however, the levels of phosphohydroxypyruvate:l-glutamate transaminase were comparable to those of d-3-phosphoglycerate dehydrogenase in a given tissue as were those for hydroxypyruvate: l-alanine transaminase and d-glycerate dehydrogenase.     

Regulation of synthesis of glutamate dehydrogenase and glutamine synthetase in micro-organisms

1. Aspergillus nidulans, Neurospora crassa and Escherichia coli were grown on media containing a range of concentrations of nitrate, or ammonia, or urea, or l-glutamate, or l-glutamine as the sole source of nitrogen and the glutamate dehydrogenate and glutamine synthetase of the cells measured. 2. Aspergillus, Neurospora and Escherichia coli cells, grown on l-glutamate or on high concentrations of ammonia or on high concentrations of urea, possessed low glutamate dehydrogenase activity compared with cells grown on other nitrogen sources. 3. Aspergillus, Neurospora and Escherichia coli cells grown on l-glutamate possessed high glutamine synthetase activity compared with cells grown on other nitrogen sources. 4. The hypothesis is proposed that in Aspergillus, Neurospora and Escherichia colil-glutamate represses the synthesis of glutamate dehydrogenase and l-glutamine represses the synthesis of glutamine synthetase. 5. A comparison of the glutamine-synthesizing activity and the gamma-glutamyltransferase activity of glutamine synthetase in Aspergillus and Neurospora gave no indication that these fungi produce different forms of glutamine synthetase when grown on ammonia or l-glutamate as nitrogen sources.     

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.     

Improvement in thermal stability of l-fucose dehydrogenase by chemical modification and additives for use as an NADPH recycling reagent

Chemical modification and addition of stabilizers were evaluated to stabilize l-fucose dehydrogenase (FDH) in mild alkaline which is required for NADPH recycling used in a glutamate dehydrogenase (GDH)-catalyzed elimination of ammonia. FDH lost all its activity at pH 9 after 10 min at 50 °C, while FDH conjugated with dextran retained about 30% activity. EDTA or citric acid, at 50–100 mM, increased the thermal stability of FDH with retention of more than 70–80% activity after heat-treatment. FDH was then functional for NADPH regeneration.