Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5''-phosphate.

The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.     

Multiple interactions of lysine-128 of Escherichia coli glutamate dehydrogenase revealed by site-directed mutagenesis studies

A highly conserved lysine at position 128 of Escherichia coli glutamate dehydrogenase (GDH) has been altered by site-directed mutagenesis of the gdhA gene. Chemical modification studies have previously shown the importance of this residue for catalytic activity. We report the properties of mutants in which lysine-128 has been changed to histidine (K128H) or arginine (K128R). Both mutants have substantially reduced catalytic centre activities and raised pH optima for activity. K128H also has increased relative activity with amino acid substrates other than glutamate, especially L-norvaline. These differences, together with alterations in Km values, Kd values for NADPH and Ki values for D-glutamate, imply that lysine-128 is intimately involved in either direct or indirect interactions with all the substrates and also in catalysis. These multiple interactions of lysine-128 explain the diverse effects of chemical modifications of the corresponding lysine in homologous GHDs. In contrast, lysine-27, another highly reactive residue in bovine GDH, is not conserved in all of the sequenced NADP-specific GDHs and is therefore not likely to be involved in catalysis.     

Modification of glutamate dehydrogenase by pyridoxal-5'-phosphate. Study of the structural organisation of the hexamer and its possible role in the realization of GTP action

The catalytic and regulator properties of glutamate dehydrogenase by modification of Lys-126 residue by puridoxal-5'-phosphate was studied. The phosphopyridoxyl derivative of the enzyme with blocked NADH-induced binding site of GTP not capable of being polymerized was taken as a model. It was shown that: blocking the epsilon-amino group of Lys-126 residue brings to a simultaneous inactivation of the enzyme and desensibilization of its residual activity to GTP action; the modification of Lys-126 residue and resulting inactivation of the enzyme and desensibilization to GTP action were non-cooperative processes, with equal values of pseudofirst order rate constants; modification of Lys-126 residue of any of hexamer's protomer results in the desensibilization to GTP action on one of the contacting, catalytically active protomers. The experimental dependence of the inhibition degree of the enzyme by GTP upon the average number of modified residues of Lys-126 is explained by the model of the hexamer of glutamate dehydrogenase with identical interlocation of any of the protomers in relation to the one in contact.     

3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.     

Physicochemical evidence for the existence of two pyridoxal 5'-phosphate binding sites on glutamate dehydrogenase and characterization of their functional role.

Kinetic studies of pyridoxal 5'-phosphate binding to glutamate dehydrogenase (EC 1.4.1.3) has provided evidence for two specific binding sites, chemically identified as Lys 126 and Lys 333. Use of protecting ligands permitted the selective modification of only one of these lysines, and showed that (1) Lys 333 modification results in depolymerisation of the enzyme into active hexamers; (2) Lys 126-modified enzyme was 92% inactivated. The residual activity was desensitized to GTP. The inactivation process was cooperative, maximum inactivation occurring as soon as half of the Lys 126 were modified.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.     

The effect of modifying lysine-126 on the physical, catalytic and regulatory properties of bovine liver glutamate dehydrogenase

1. The reaction of 4-iodoacetamidosalicylate with bovine liver glutamate dehydrogenase is dependent on pH. The pH-activity curve is bell-shaped and can be described by apparent pK values of 7.8+/-0.2 and 9.1+/-0.2. 2. Enzyme in which lysine-126 has been modified by 4-iodoacetamidosalicylate has unaltered sedimentation characteristics except when measured in the presence of GTP and NADH. 3. GTP binding to the inhibited enzyme is unaltered. However, GTP can no longer promote the binding of a second molecule of NADH, since this is already bound to the inhibited enzyme without GTP. 4. The equilibrium binding of ADP, GTP, NAD-sulphite and NADH (when measured at low concentrations) was largely unchanged by modification. 5. The number of binding sites for 2-oxoglutarate to the enzyme-NADH complex were decreased by 60% in an enzyme that has been inhibited by 70%.     

Studies of glutamate dehydrogenase. Analysis of quaternary structure and contact areas between the polypeptide chains

Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (molecular weight 168000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with mono-functional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino-terminal residue alanine-1 and the others involve lysine-105, lysine-154, lysine-269, lysine-358 and lysine-399. Quantitative analysis of the specific radioactivity of each peptide/mol lysine leads to the conclusion that only lysine-105, lysine-154, lysine-269 and lysine-358 participate in cross-links, lysine-269 and lysine-358, respectively, being at isologous and lysine-105 cross-linked with lysine-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains.     

Reactive cysteine residue of bovine brain glutamate dehydrogenase isoproteins

Protein chemical studies of glutamate dehydrogenase isoproteins (GDH I and GDH II) from bovine brain reveal that one cystein residue is accessible for reaction with thiol-modifying reagent. Reaction of the two types of GDH isoproteins with p-chloromercuribenzoic acid resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order kinetics with the second-order rate constant of 83 M(-1) s(-1) and 75 M(-1) s(-1) for GDH I and GDH II, respectively. The inactivation was partially prevented by preincubation of the glutamate dehydrogenase isoproteins with NADH. A combination of 10 mM 2-oxoglutarate with 2 mM NADH gave complete protection against the inactivation. There were no significant differences between the two glutamate dehydrogenase isoproteins in their sensitivities to inactivation by p-chloromercuribenzoic indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Allosteric effectors such as ADP and GTP had no effects on the inactivation of glutamate dehydrogenase isoproteins by thiol-modifying reagents. By a combination of peptide mapping analysis and labeling with [14C] p-chloromercuribenzoic acid, a reactive cystein residue was identified as Cys323 in the overall sequence. The cysteine residue was clearly identical to sequences of other GDH species known.     

Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver

Photooxidation of bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3) in the presence of methylene blue at a low light intensity occurs in two stages. At the first stage, the duration of which depends on temperature and dye concentration, a slight activation is observed simultaneously with the oxidation of two histidine residues. At the second stage, the inactivation is concomitant with the oxidation of three histidine and one tryptophan residues. The inactivation is a first order reaction (k = 3,22 X 10(-2) min-1) and is correlated with changes in the circular dichroism spectra. These data testify to the structural role of histidine residues in the GDH molecule. The kinetic behaviour of GDH during its modification with diethylpyrocarbonate (DEP) depends on pH and the reagent concentration. Four histidine residues undergo carbethoxylation at pH 6.0 and 7.5, but the modification rate is much higher at pH 7.5. At low DEP concentrations, a remarkable activation is observed with a simultaneous modification of one histidine residue, which is independent of pH. At high DEP concentrations, a rapid inactivation takes place at pH 7.5. Treatment of the carbethoxylated inactive enzyme with hydroxylamine results in the deacylation of histidine residues without any noticeable reactivation. The data on the combined effect of DEP and pyridoxal-5'-phosphate suggest that GDH inactivation by DEP at pH 7.5 is a result of modification of an essential epsilon-NH2 group of lysine-126.     

Haemonchus contortus: enzymes. 3. Glutamate dehydrogenase

Adult male and female Haemonchus contortus were homogenized and subjected to differential centrifugation. The crude, high-speed, supernatant fraction contained more than 95% of the glutamate dehydrogenase activity. The enzyme was purified through use of DEAE-cellulose columns and sucrose density gradient centrifugation. The enzyme from both crude and purified preparations was detected as a single band of activity following starch or polyacrylamide-gel electrophoresis. The Haemonchus enzyme was compared with ovine and bovine liver glutamate dehydrogenases. The three enzymes were similar in molecular size, Michaelis constants, and pH optimums but differed in electrophoretic mobility in polyacrylamide-gels, activity with NADP as coenzyme, and effect of AMP and ADP on activity. Sheep anti-Haemonchus glutamate dehydrogenase serum inhibited Haemonchus glutamate dehydrogenase, but did not inhibit the ovine or bovine enzymes.     

The equilibrium position of the reaction of bovine liver glutamate dehydrogenase with pyridoxal5''-phosphate. A demonstration that covalent modification with this reagent completely abolishes catalytic activity.

1. The activity of bovine liver glutamate dehydrogenase incubated with pyridoxal 5'-phosphate declined to a steady value reached within 30--60 min. The residual activity depended on the concentration of modifier up to about 5 mM. Above this concentration, however, no further inactivation was produced. The minimum activity obtainable in such incubations was 6--7% of the initial value. 2. Km values of the modified enzyme were unaltered, whereas Vmax. was decreased. 3. Activity was fully regained on dialysis against 0.1 M-potassium phosphate buffer. 4. Reduction with borohydride rendered the inactivation permanent but did not alter its extent. 5. Enzyme permanently inactivated in this way to the extent of 90% and dialysed was re-treated with pyridoxal 5'-phosphate. In this second cycle activity declined from 10 to 1% of the original activity. 6. This strongly suggests that the failure to achieve complete inactivation in a single cycle reflects a reversible equilibrium between inactive Schiff base, i.e. covalently modified enzyme, and a non-covalent complex. 7. The re-inactivation reaction occurring on dilution was demonstrated directly and a first-order rate constant obtained (0.048 min-1). This, in conjunction with an estimate of the forward rate constant for Schiff-base formation, obtained by approximate pseudo-first-order analysis of inactivation at varied modifier concentrations, gives a predicted minimum activity very close to that actually obtained in a single cycle of treatment. 8. The dissociation constant of the non-covalent complex is given by two methods as 0.90 and 1.59mM. 9. The results indicate that covalent modification with pyridoxal 5'-phosphate completely abolishes the activity of glutamate dehydrogenase.     

Structural organization of glutamate dehydrogenase. 2. Inactivation and dissociation of immobilized hexamer induced by urea

The urea-induced inactivation and dissociation of catalytically active hexamer of glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) from bovine liver were studied using radioactive phosphopyridoxyl derivative of the enzyme immobilized on cyanogen bromide-activated Sepharose CL-4B. It is shown that at neutral pH (7.0-7.8) urea causes dissociation of glutamate dehydrogenase to directly yield catalytically inactive immobilized monomers rather than hexamer's stable fragments at the same time. At pH 8.9 or 5.6 the urea-induced is accompanied by the formation of conformationally stable immobilized dimers or trimers, respectively. The trimers are catalytically active, whereas the dimers did not exhibit any enzymatic activity. The data obtained led to suggestion that the hexamer consists of three either equivalent dimers (3 alpha 2) or of two equivalent trimers (2 alpha 3).     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. IV. The COOH-terminal 669 residues of the peptide chain; comparison with other glutamate dehydrogenases.

A sequence is presented for the COOH-terminal 669 residues of the NAD-specific glutamate dehydrogenase of Neurospora crassa. Comparison of this sequence with those of the vertebrate glutamate dehydrogenases of chicken and bovine liver and with the NADP-specific enzyme of Neurospora shows some similarities in sequences around residues previously identified as important for the function of these enzymes. These are: (a) the reactive lysine residue of low pK in the NADP and the vertebrate enzymes; (b) the tyrosine residue of the NADP enzyme that is readily nitrated by tetranitromethane with inactivation, a residue protected by NADP or by NMN; and (c) the arginine residue of the NADP-enzyme that is reactive with 1,2-cyclohexanedione with inactivation. Despite these similarities, comparison of the sequence of the NAD-enzyme with those of the other glutamate dehydrogenases of known sequences revealed relatively little overall homology as determined by computer analysis.     

Cooperative inactivation of glutamate dehydrogenase of 2,2,6,6- tetramethyl-4-oxopiperidine-1-oxyl. Interpretation of results within the scope of a hexamer model with equivalent subunit orientation

It was shown that the blockage of epsilon-amino group of Lis-126 residue by 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl (TMPO) leads to the cooperative inactivation of glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3). The data concerning cooperative inactivation of the enzyme are interpreted by the model of hexamer with identical orientation of subunits. It was shown that the modification of any of enzyme subunits is accompanied by an inactivation of the hexamer's fragment which is a dimer, with subunits interacting reciprocally by means of isological contacts.     

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5''-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.     

Effect of temperature on the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases

A study on the response of the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases to temperature variations has been carried out. The thermodynamic properties of the heat inactivation process and of the reaction with the substrates glutamate and α-ketoglutarate have been investigated. The heat inactivation of nuclear glutamate dehydrogenase proceeds at a faster rate than that of the mitochondrial enzyme in the temperature range 40–51 °C; the enthalpy of activation of the inactivation process is higher and the entropy is almost double, compared to the values of mitochondrial glutamate dehydrogenase. The effect of temperature on the maximal velocity shows that, with both glutamate and α-ketoglutarate, the enthalpy of activation with nuclear glutamate dehydrogenase is double and the decrease in entropy almost half of the values of the mitochondrial enzyme. The variation of the apparent K_m with temperature shows a decrease of the affinity of both enzymes for glutamate, with no major difference in the thermodynamic properties of the reaction. With α-ketoglutarate, on the other hand, the affinity of nuclear glutamate dehydrogenase decreased, whereas that of the mitochondrial enzyme increased with temperature. The process is therefore exothermic with the former enzyme, endothermic with the latter; furthermore, it occurs with a decrease in enthropy with nuclear glutamate dehydrogenase, but with a large increase with the mitochondrial enzyme. The studies on the effect of temperature on the activity were carried out in the range 20–44 °C.     

Functional arginine residues involved in coenzyme binding by glutamate dehydrogenases.

Reaction of the NADP-dependent glutamate dehydrogenase of Neurospora with 1,2-cyclohexanedione results in a biphasic loss of enzyme activity. At the end of the rapid phase of the reaction (t1/2 = 1.5 min) the enzyme activity is diminished by approximately 60% with the simultaneous loss of 1 residue of arginine per subunit. After 60 min, the enzyme activity is completely lost with the modification of a total of 2 arginine residues per subunit. Reaction of bovine liver glutamate dehydrogenase with cyclohexanedione causes a rapid loss of approximately 45% of the enzyme activity and modification of about 1.5 residues of arginine per subunit. More prolonged treatment results in reaction of an additional 4 residues of arginine per subunit but is without further effect on the residual activity. The activity of the Neurospora enzyme is not protected by substrate, coenzyme, or a combination of both; however, the activity of the bovine enzyme is partially protected by high levels of NAD or NADP. Although the Km for alpha-ketoglutarate is unchanged by a limited modification of either enzyme with cyclohexanedione, the Km for coenzyme is increased about 2-fold for the Neurospora enzyme and about 1.5-fold for the bovine enzyme. The Ki of the Neurospora dehydrogenase for the competitive inhibitor 2'-monophosphoadenosine-5'-diphosphoribose is unchanged by the enzyme modification, but nicotinamide mononucleotide, a competitive inhibitor for the native Neurospora enzyme, does not inhibit the glutamate dehydrogenase with 1 modified arginine residue. This finding implies that the modified arginine is at or near the nicotinamide binding iste of the enzyme.     

Phosphorylation of NAD-dependent glutamate dehydrogenase from yeast.

The NAD-dependent glutamate dehydrogenase from Candida utilis was isolated from 32P-labeled cells following enzyme inactivation promoted by glutamate starvation and found to exist in a phosphorylated form. Analysis of purified, fully active NAD-dependent glutamate dehydrogenase (a form) and inactive NAD-dependent glutamate dehydrogenase (b form) for alkalilabile phosphate revealed that the a form contained 0.09 +/- 0.06 mol of phosphate/mol of enzyme subunit and b form 1.25 +/- 0.06 mol of phosphate/mol of enzyme subunit. Phosphorylation caused a 10-fold reduction in enzyme specific activity. Dephosphorylation (release of 32P) and enzyme reactivation occurred on incubation with cell-free yeast extracts, indicating the presence of a phosphoprotein phosphatase in such preparations.     

Mitochondrial and nuclear glutamate dehydrogenases in Chinese hamster ovary cells in culture.

Nuclear glutamate dehydrogenase (EC 1.4.1.3) activity has been demonstrated in Chinese hamster ovary cells. Some characteristics of this enzyme have been examined and compared with those of the mitochondrial glutamate dehydrogenase from the same source. Differences were detected in the extent of the activation by inorganic phosphate, in the pH versus activity curves, in the affinity of the two enzymes for the cofactor NAD+ and in the electrophosretic mobility. A different rate of decay of the two enzymes has been observed in cells grown in the presence of chloramphenicol. Immunological studies show that, as in ox liver, the nuclear enzyme has specific antigenic determinants besides those in common with mitochondrial glutamate dehydrogenase. Finally, experiments of thermal inactivation indicate a higher stability of the mitochondrial enzyme.