Native Glutamate Dehydrogenase Research Articles

Biochemical and Epigenetic Regulation of Glutamate Metabolism in Maize (Zea mays L.) Leaves under Salt Stress.

The effect of salt stress (150 mM NaCl) on the expression of genes, methylation of their promoters, and enzymatic activity of glutamate dehydrogenase (GDH), glutamate decarboxylase (GAD), and the 2-oxoglutarate (2-OG)-dehydrogenase (2-OGDH) complex was studied in maize (Zea mays L.). GDH activity increased continuously under salt stress, being 3-fold higher after 24 h. This was accompanied by the appearance of a second isoform with lower electrophoretic mobility. The expression of the Gdh1 gene strongly increased after 6-12 h of incubation, which corresponded to the demethylation of its promoter, while Gdh2 gene expression slightly increased after 2-6 h and then decreased. GAD activity gradually increased in the first 12 h, and then returned to the control level. This corresponded to the increase of Gad expression and its demethylation. Salt stress led to a 2-fold increase in the activity of 2-OGDH during the first 6 h of NaCl treatment, then the activity returned to the control level. Expression of the genes Ogdh1 and Ogdh3 peaked after 1-2 h of incubation. After 6-8 h with NaCl, the expression of these genes declined below the control levels, which correlated with the higher methylation of their promoters. We conclude that salt stress causes a redirection of the 2-OG flux to the γ-aminobutyric acid shunt via its amination to glutamate, by altering the expression of the Gdh1 and Gdh2 genes, which likely promotes the assembly of the native GDH molecule having a different subunit composition and greater affinity for 2-OG.

Mechanisms of activity loss for a multi-PEGylated protein by experiment and simulation

Over the past five decades, one of the most investigated strategies to increase the circulatory half-life and to reduce the immunogenicity of non-human therapeutic proteins has been to cover their surface with multiple copies of methoxy poly(ethylene glycol) (mPEG). This multi-‘PEGylation’ strategy, however, is often associated with loss of protein activity, which is most often rationalized as direct modification of the protein's active site or to steric hindrance near its surface. While these mechanisms are generally accepted and likely valid, very few studies investigate other possible mechanisms of activity loss. To answer this question, this study investigates the mechanisms of activity loss for glutamate dehydrogenase (GDH) bearing up to 25 chains of mPEG per protein. Through a combination of experimental design and simulation, the most common proposed causes of protein inactivation (i.e. active-site PEGylation and steric hindrance near the surface of the protein) are circumvented to shed light on less characterized mechanisms such as altered protein structure, altered microenvironment at the surface of the protein, and altered protein dynamics. Experiments suggest that the secondary and tertiary structure of GDH was (sometimes significantly) affected by PEGylation, though these changes do not necessarily result in a loss of activity. Structure-activity correlations could not reconcile all trends within the library of bioconjugates tested. Moreover, by measuring catalytic activity in the presence of five different allosteric modulators, loss of activity could not be ascribed to an altered local microenvironment at the protein's surface. As such, coarse-grained simulations were exploited to reveal the effect of PEGylation on protein dynamics, which are long-ranged, cooperative, and essential for GDH catalytic activity. Normal mode analysis of native bovine GDH (i.e. not PEGylated) revealed that the lowest ranking mode displayed several features that bore similarity to movements known for the native protein. These included rotation/distortion of the ‘antenna’ regions, closure of the ‘catalytic cleft’, and constriction of the internal cavity. A reduced magnitude of this mode was observed as a function of degree of PEGylation and mPEG molecular weight and correlated well with trends observed for the library of bioconjugates tested experimentally. Overall, this study provides new insight into fundamental considerations associated with loss of activity, which will contribute to the design of future bioconjugates by providing a better understanding of possible mechanisms of loss of activity.

Purification and Side Chain Selective Chemical Modifications of Glutamate Dehydrogenase from Bacillus subtilis Natto

Glutamate dehydrogenase (GDH) from Bacillus subtilis natto was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, size exclusion chromatography, and hydroxyapatite (HA) affinity chromatography. The GDH was purified 34-fold, with a yield of 41% of total activity and a specific activity of 34.29U/mg proteins. The molecular weight (Mr) of was measured at 47kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 264kDa with high-performance liquid chromatography (HPLC). The optimum pH and temperature for the deammoniation reaction were measured to be 7.5 and 30°C, respectively. The active-site amino acid residues of GDH were investigated by chemical modification. The compounds 2,4,6-trinitrobenzenesulfonic acid (TNBS), phenylglyoxal (PG), and phenylmethanesulfonyl fluoride (PMSF) were used to modify lysine, arginine, and serine active site residues, respectively. After treatment with modifying reagents at concentrations of 1mM, GDH activity fell to 10.7% with TNBS, 83.3% with PG, and 12.8% with PMSF. However, with substrate protection, there was almost no loss in GDH activity following treatment with any modifying reagent. The kinetic parameters K m and V max were determined in each case. K m values for native GDH, 50% TNBS-inactivated GDH, and 50% PMSF-inactivated GDH were 0.037, 0.104, and 0.017mM, respectively. V max values were 0.048, 0.022, and 0.031mM/s, respectively. These results suggest that the active site contains one or more lysine residues that play a role in substrate binding and one or more serine residues that may maintain the enzyme conformation. However, arginine residues played less of a role in the activity of GDH.

Characterization of native glutamate dehydrogenase from an aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285 +/- 15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 degrees C). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7-15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.

Limited proteolysis of glutamate dehydrogenase in the liver of eel ( Anguilla japonica)

1. 1. Limited proteolyzed glutamate dehydrogenase (GDH) was purified from acetone powder prepared from fresh eel livers. 2. 2. The limited proteolyzed GDH has a subunit of 50,000 molecular weight as well as a subunit of 54,000 molecular weight, and the specific activity of the GDH was higher than that of the native GDH. 3. 3. The amino acid composition and sequence of four residues from the N- terminal of the limited proteolyzed GDH were the same as those of the trypsin-treated GDH (Tang et al., J. Biochem. 111, 655–661, 1992). 4. 4. Effects of PCMB, ADP and GTP on the limited proteolyzed GDH were comparable with those on the trypsin-treated GDH. 5. 5. Proteases extracted from the acetone powder of the fresh livers were applied to a typsin inhibitor-Sepharose column, and proteases retained on this column did not bring about limited proteolysis of the native GDH.

The trypsin-catalyzed activation of glutamate dehydrogenase purified from eel liver.

Eel liver glutamate dehydrogenase (GDH) [EC 1.4.1.3] was eightfold activated by trypsin and the molecular weight of the subunit of the native GDH decreased from 54,000 to 50,000. The C-terminal amino acid of both subunits was Thr. One peptide was released after proteolysis of the native GDH by trypsin and purified by anhydrotrypsin agarose and reversed-phase HPLC. The isolated peptide consisted of 39 amino acids and its amino acid sequence was as follows: H2NS-E-A-V-E-K-E-D-D-P-N-F-F-K-M-V-E-G-F-F-D-K-G-A-A-I- V-E-N-K-L-V-E-E-D-L-K-T-R-COOH. The peptide contained the N-terminal of the native GDH and its molecular weight was calculated to be 4,413. We concluded that the trypsin-catalyzed activation was caused by release of this peptide from the native GDH. p-Chloromercuribenzoic acid inhibited the activity of the trypsin-treated GDH, but stimulated that of the native GDH. The response of trypsin-treated GDH to ADP and GTP was decreased compared with that of the native GDH.

Affinity labeling of an allosteric ADP site of glutamate dehydrogenase by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate.

Bovine liver glutamate dehydrogenase reacts covalently with the adenine nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of about 1 mol of reagent/mol of enzyme subunit. The modified enzyme is not inactivated by this reaction as measured in the absence of allosteric effectors. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of NADH; both of these effects are irreversibly decreased upon reaction of the enzyme with 2-BDB-TAMP. The decrease in activation by ADP was used to determine the rate constant for reaction with 2-BDB-TAMP. The rate constant (kobs) for loss of ADP activation exhibits a nonlinear dependence on 2-BDB-TAMP concentration, suggesting a reversible binding of reagent (KR = 0.74 mM) prior to irreversible modification. At 1.2 mM 2-BDB-TAMP, kobs = 0.060 min-1 and is not affected by alpha-ketoglutarate or GTP, but is decreased to 0.020 min-1 by 5 mM NADH and to zero by 5 mM ADP. Incorporation after incubation with 1.2 mM 2-BDB-TAMP for 1 h at pH 7.1 is 1.02 mol/mol enzyme subunit in the absence but only 0.09 mol/subunit in the presence of ADP. The enzyme protected with 5 mM ADP behaves like native enzyme in its activation by ADP and in its inhibition by NADH. Native enzyme binds reversibly 2 mol of [14C]ADP/subunit, whereas modified enzyme binds only 1 mol of ADP/peptide chain. These results indicate that incorporation of 1 mol of 2-BDB-TAMP causes elimination of one of the ADP sites of the native enzyme. 2-BDB-TAMP acts as an affinity label of an ADP site of glutamate dehydrogenase and indirectly influences the NADH inhibitory site.

Magnetic-resonance studies of the geometry of bound substrate, coenzyme and activator on bovine-liver glutamate dehydrogenase.

ADP and ATP with a spin-label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin-label at a site in the active centre[Andree and Zantema, (1978) Biochemistry, 17, 778--783]. The spin-labelled activators interact with ketone spin-labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin-spin interaction between the ketone spin-label and the spin-labelled activators is observed. Nuclear magnetic resonance studies of the linewidth of 2-oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin-labels result in distances between the ligand nuclei and the spin-labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with the coenzyme site.

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.

Affinity labeling of a regulatory site of bovine liver glutamate dehydrogenase.

A new adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), has been synthesized which reacts covalently with bovine liver glutamate dehydrogenase. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of DPNH. Both of these effects are irreversibly decreased upon incubation of the enzyme with the adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), while the intrinsic enzymatic activity as measured in the absence of regulatory compounds remains unaltered. A plot of the rate constant for modification as a function of the 3'-FSBA concentration is not linear, suggesting that the adenosine derivative binds to the enzyme (Ki equals 1.0 times 10-4 M) prior to the irreversible modification. Protection against modification by 3'-FSBA is provided by ADP and by high concentrations of DPNH, but not by the inhibitor GTP, the substrate alpha-keto glutarate, the coenzyme TPNH, or low concentrations of the coenzyme DPNH. The isolated altered enzyme contains approximately 1 mol of sulfonylbenzoyladenosine per peptide chain, indicating that a single specific regulatory site has reacted with 3'-tfsba. the modified enzyme exhibits normal Michaelis constants for its substrates and is still inhibited by GTP, albeit at a higher concentration, but it is not inhibited by high concentrations of DPNH. Although ADP does not appreciably activate the modified enzyme, it does (as in the case of the native enzyme) overcome the inhibition of the modified enzyme by GTP. These results suggest that ADP can bind to the modified enzyme, but that its ability to activate is affected indirectly by the modification of the adjacent tdpnh inhibitory site. It is proposed that the regulatory sites for ADP and DPNH are partially overlapping and that 3'FSBA functions as a specific affinity label for the DPNH inhibitory site of glutamate dehydrogenase. It is anticipated that 3'-p-fluorosulfonylbenzolyadenosine may act as an affinity label of other dehydrogenases as well as of other classes of enzymes which use adenine nucleotides as substrates or regulators.

Glutamate Dehydrogenase Concentration as a Determinant in the Effect of Purine Nucleotides on Enzymatic Activity

Abstract The properties of bovine liver glutamate dehydrogenase with respect to the effects of purine nucleotides have been examined over a wide range of enzyme concentrations. Kinetic studies, with enzyme concentration up to 1 mg per ml have been performed with both guanosine triphosphate and diphosphate as inhibitors and adenosine diphosphate as an activator of the enzyme. Direct binding experiments have been performed to examine GTP binding in the presence of reduced diphosphopyridine nucleotide over the range of 0.1 to 14.7 mg per ml. Both the kinetic and direct binding studies support the conclusion that, in the presence of coenzyme, GTP and GDP bind less tightly to the associated polymeric form (which has a molecular weight of 1.6 to 2 x 106 and is formed as a result of a concentration-dependent association reaction) than they do to the monomeric form of the enzyme (molecular weight, 4 x 105). The preferential binding of these nucleotides to monomer gives rise to an apparent cooperative interaction between inhibitor sites (either GTP or GDP) at enzyme concentrations greater than 0.1 mg per ml. No such effects exist at lower enzyme levels (l0.01 mg per ml), presumably because no polymer is present. In contrast to these results, ADP, which activates the reaction, has been found in the presence of reduced pyridine nucleotide to bind preferentially to the polymer rather than to the monomer. It is postulated that DPNII and reduced triphosphopyridine nucleotide alone behave in the same manner as ADP. Experiments have been performed to evaluate the effect of both ADP and GTP (or GDP) simultaneously on the kinetic properties at high enzyme concentrations. Kinetic data and theory are presented for the situation where the enzyme concentration is larger than the dissociation constant for one of the ligands (that is, where the free and total ligand concentration cannot be considered to be equivalent) as is the case for inhibition by GTP. The theory is shown to be valid for a chemically modified glutamate dehydrogenase which is enzymatically normal in all respects but which cannot undergo association hat high enzyme concentrations. The theory presented here, however, is not adequate for native glutamate dehydrogenase presumably because of the preferential binding of GTP to the monomeric form of the enzyme. The consequence of preferential binding of purine nucleotides to different molecular weight species is discussed. It is noted that the ability of an enzyme to undergo association may be an important factor in relation to the effect of substrate or allosteric ligands on enzyme activity. Finally, it is shown by stop flow experiments that the specific activity of the enzyme in the absence of purine nucleotides is essentially independent of enzyme concentration.

A viscosity and sedimentation study on glutamate dehydrogenase

Summary The viscosity of native glutamate dehydrogenase ( L -glutamate:NAD(P)-oxidoreductase (deaminating), EC 1.4.1.3) has been determined in the presence and the absence of small molecules known to affect its aggregation properties. The effects on viscosity of denaturing agents such as sodium dodecyl sulfate and 8 M urea were also studied. Approximate molecular weights from sedimentation and viscosity data were computed for the native enzyme and some of its subunits.