Bovine Liver Glutamate Dehydrogenase Research Articles

A chromophoric and fluorescent analog of GTP, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP, as a spectroscopic probe for the GTP inhibitory site of liver glutamate dehydrogenase.

The ribose-modified chromophoric and fluorescent analog of ATP, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP (TNP-ATP) (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635-647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293-297) has been widely used as an ATP analog for various ATPases. Although the corresponding analog of GTP,2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP (TNP-GTP) should be useful for the study of various GTP-requiring enzymes, it is difficult to prepare TNP-GTP by the conventional method. In the present study, we succeeded in the synthesis of TNP-GTP with the use of an alternative method. The analogs of GDP, GMP, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) were also synthesized. Visible absorption and fluorescent properties of TNP-GTP, TNP-GDP, TNP-GMP, and TNP-Gpp(NH)p were quite similar to those of TNP-ATP. TNP-GTP was found to be able to replace GTP as an inhibitor for bovine liver glutamate dehydrogenase. The enzyme was inhibited by TNP-GTP to a maximum extent of 54% at saturating concentrations of the analog with a KI of 2.7 microM. TNP-Gpp(NH)p and other ribose-modified fluorescent analogs of GTP,3'-O-anthraniloyl-GTP and 3'-O-(N-methylanthraniloyl)-GTP (Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508), also inhibited the enzymatic activity. Binding of TNP-GTP to the enzyme was characterized by a 5.6-fold enhancement in analog fluorescence. In the presence of NADH, the limiting fluorescence enhancement of the bound analog decreased to 2.7-fold. As determined by fluorometric titration, the maximum number of TNP-GTP binding sites on the enzyme was 1.9 mol/mol of subunit with a KD of 0.66 microM in the absence of NADH and 2.2 mol/mol of subunit with two KD values of 0.11 and 0.71 microM in the presence of NADH. These observations suggest that NADH binding increases the affinity of only 1 mol of the 2 mol of TNP-GTP bound to the enzyme. These spectroscopic and biological properties of TNP-GTP should make this analog useful as a chromophoric and fluorescent probe for studies not only of glutamate dehydrogenase but also of various GTP-requiring enzymes, which have a high specificity for the base moiety of GTP.

Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase.

5'-p-Fluorosulfonylbenzoyladenosine (5'-FSBA) is a specific affinity label for the inhibitory NADH site of bovine liver glutamate dehydrogenase. Reaction of the enzyme with 5'-FSBA results in the loss of inhibition by high concentrations of NADH with covalent attachment of 0.53 sulfonylbenozyladenosine/subunit, i.e. modification of three subunits of the hexameric enzyme. Equal amounts of N epsilon-(4-carboxybenzenesulfonyl)lysine (Lys-(CBS] and O-(4-carboxybenzenesulfonyl)tyrosine (Tyr-(CBS] are found throughout the course of the reaction (Saradambal, K. V., Bednar, R. A., and Colman, R. F. (1981) J. Biol. Chem. 256, 11866-11872). Modified enzyme, prepared by incubating 2 mg/ml glutamate dehydrogenase with 0.3 mM 3H-labeled 5'-FSBA at pH 8 for 1 h, was carboxymethylated and digested with thermolysin. Two nucleosidyl peptides were isolated by a combination of chromatography on phenyl boronate-agarose, high-performance liquid chromatography in ammonium bicarbonate and high-performance liquid chromatography in trifluoroacetic acid. By comparison of the amino acid analysis and NH2-terminal residue of each isolated peptide with the known amino acid sequence of the enzyme, the peptides were identified as Leu-Gly-Arg-Lys(CBS) and Ile-Gly-His-Tyr(CBS)-Asp. These sequences correspond to residues 417-420 and 187-191, respectively. Lys-420 and Tyr-190 of glutamate dehydrogenase react with 5'-FSBA, and both are apparently located in the NADH inhibitory site.

Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate.

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody to glutamate dehydrogenase isolation and characterization

This paper describes tne isolation and characterization of a monoclonal antibody to bovine liver glutamate dehydrogenase (GDH). Monoclonal antibody is mouse immunoglobulin subclass IgG 2a and reacts strongly with the antigen in an enzyme-linked immunosorbent assay (E-LISA). Its specificity was determined by an antigen binding assay and by Western blotting. Potential uses and possible applications are discussed.

Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.

The binding of 1, N6-etheno-nad to bovine liver glutamate dehydrogenase

The binding of 1, N 6-etheno-NAD (ϵNAD) to bovine liver glutamate dehydrogenase ( l-glutamate;NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate has been investigated at pH 7.0, 0.05 M phosphate buffer at 20°C, by fluorescence titrations. ϵNAD binds to the protein in a simple fashion: one molecule of coenzyme per enzyme polypeptide chain in the range of enzyme concentrations investigated (from above 50 to a few micromoles of enzyme polypeptide chains/liter). The fluorescence enhancement factor, Q, of bound ϵNAD relative to free ϵNAD is independent of the saturation degree, as deduced from the constant value of the long fluorescence decay lifetime (about 21 ns), and is about 17, as deduced from F max/ F 0 ratio values obtained after extrapolation from double reciprocal plots of 1/Δ F vs. 1/[glutamate dehydrogenase]. This value for the Q factor is also independent of enzyme concentration, as well as of the presence of either GTP or ADP. At low enzyme concentrations (below 20 μM polypeptide chains/liter), the dissociation constant of ϵNAD increases progressively from a plateau value of about 50 μM to about 100 μM at infinite dilution. This is interpreted as being due to a minor affinity of glutamate dehydrogenase hexamers, with respect to higher aggregation states of the enzyme, towards ϵNAD. As expected, GTP and ADP change the affinity of glutamate dehydrogenase towards ϵNAD in an opposite manner: GTP strongly increases it, whereas ADP strongly decreases it ( K d app around 6 μM with saturating GTP and around 300 μM with saturating ADP). Furthermore, in the case of GTP, both GTP and ϵNAD bind to glutamate dehydrogenase with positive cooperativity, with a Hill coefficient of approx. 1.8 for both and a K d app ≈ 30 μM for the binding of GTP to glutamate dehydrogenase saturated with ϵNAD and glutarate. The value of the Q factor remains the same, even in the presence of the effectors (again from lifetime measurements), as well as the number of ϵNAD binding sites per enzyme polypeptide chain. These results are interpreted in terms of independent active sites, in the case without effectors. With ADP the binding appears to be simple, but no careful investigation has been attempted at low enzyme concentrations because of the low saturation degree achievable, whereas with GTP the cooperativity can be explained as due to a shift towards hexamers from higher aggregation states.

Light-scattering study on subunit association-dissociation equilibria of bovine liver glutamate dehydrogenase

The subunit dissociation of bovine liver glutamate dehydrogenase ( l-glutamate: NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3) induced by guanidine hydrochloride (GdnHC1) in 0.2 M phosphate buffer (pH 7.3) was investigated by light-scattering molecular-weight measurements. With increasing GdnHCl concentration, two-step transition was observed in the molecular weight change. The dissociation behavior was well described by assuming the dissociation-association equilibria expressed as ▪ where H, T, and M represent the hexameric, trimeric and monomeric forms of the enzyme, respectively. GdnHCl concentration dependence of the two equilibrium constants was interpreted in terms of the binding of GdnHCl on the protein. According to this treatment, the numbers of amino acid residues present at the trimer-trimer contact area within hexamer, N 3, and at the monomer-monomer contact area within trimer, N 1, were estimated to be as follows: N 3 = 21 ± 2 and N 1 = 27 ± 5. These values seem to be reasonable considering the physical model proposed for this enzyme.

Equilibrium Kinetic Studies of Enzyme Mechanism and Control

This chapter discusses equilibrium kinetic studies of enzyme mechanism and control. It focuses on some aspects of the isotopic exchange kinetics at equilibrium and its evolution from the study of enzymic catalysis by the manipulation of reactants to the study of the control of catalysis by ligands that are not, themselves, catalytically transformed, inhibitors and activators, particularly of allosteric enzymes. The chapter presents particular areas of usefulness of these measurements with some examples. An important theroretical use of isotopic exchange at equilibrium was suggested to be the elucidation of compulsory pathways or binding orders. The usefulness of isotopic exchange kinetics for elucidation of compulsory binding orders was first established for the bovine heart and rabbit muscle lactate dehydrogenases. These enzymes were believed to have a compulsory pathway on the basis of binding and kinetic data. Subsequently, a compulsory pathway was demonstrated for porcine and bovine heart malate dehydrogenases and bovine liver glutamate dehydrogenase at pH 8.8. The study of enzyme catalysis at equilibrium by isotopic exchange of reactants is useful for elucidation of aspects of enzyme mechanism which may not be readily obtainable by other means. These include elucidation of a compulsory binding order, indications of fast and slow dissociation steps and whether chemical transformation is rate limiting; determination of the possibility of abortive complex formation, and, importantly, the observation of non-rate-limiting steps in catalysis. Another important aspect of equilibrium kinetics is its extension to the study of the mechanism of modifiers of enzymic catalysis, particularly with respect to effectors of allosteric enzymes.

Chymotryptic activation of glutamate dehydrogenase

Treatment of bovine liver glutamate dehydrogenase ( l-glutamate:NAD(P) + oxidoreductate (deaminating), EC 1.4.1.3) with chymotrypsin generates a proteolytic derivative that is activated 3–4-fold over the native enzyme. Stable preparations of activated enzyme show altered kinetic parameters (5-fold increase in K m for glutamate, 3-fold incease in V max) and altered response to allosteric modulation by GTP ad ADP. The proteolysed enzyme was less responsive to GTP and was no longer activated by ADP, although ADP binding sites remained intact on the enzyme. The effect of ADP upon substrate inhibition was altered and negative homotropic interactions in coenzyme binding were abolished.

The inhibition of glutamate dehydrogenase by derivatives of isophthalic acid

A range of compounds, structurally related to glutamate, have been tested as inhibitors of pea leaf glutamate dehydrogenase assayed in either direction. Only 5- N-substituted derivatives of aminoisophthalic acid completely inhibited the enzyme when tested at concentrations equal to either those of 2-oxoglutarate or glutamate. A minimum of three carbon atoms attached linearly to the amino group was required for maximum inhibition, inhibition was removed if there was any substitution on the first carbon. The 5- N-substituted derivatives also inhibited yeast (to a greater extent) and bovine liver (to a lesser extent) glutamate dehydrogenases.

Effect of pH on the kinetics of bovine liver glutamate dehydrogenase self-association

The temperature-jump technique with light-scattering detection was applied to the kinetic study on the self-association of bovine liver glutamate dehydrogenase (EC 1.4.1.3). The measurements were carried out for systems in phosphate buffer of differing pH, 6.3–7.8, and of differing phosphate concentration at 11.5°C. The observed association rate constant decreased with increasing phosphate concentration at the respective pH values. This observation was interpreted in terms of the inhibition from self-association by specific binding of phosphate ion to the protein. According to this interpretation, the intrinsic association rate constant, k a 0, and the binding constant of phosphate ion, K p , were estimated, k a 0 varied with pH in a manner as described by a titration curve with p K of about 7, while Kp remained almost constant in the pH range studied.

Specific modification of a single cysteine residue in both bovine liver glutamate dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase Difference in the mode of midification by pyrene maleimide

Pyrene maleimide is shown to be a ‘half of the sites’ reagent for glutamate dehydrogenase and for glyceraidehyde-3-phosphate dehydrogenase. The modified residues are identified as cysteine-115 for glutamate dehydrogenase and cysteine-149 for glyceraldehyde-3-phosphate dehydrogenase. The two enzymes react differently with pyrene maleimide. Whereas the hydrophobic environment of cysteine-115 directs the modification of glutamate dehydrogenase, the high reactivity of cysteine-149 determines the specific modification of glyceraldehyde-3-phosphate dehydrogenase. Glutamate dehydrogenase activity is unaltered by the modification; glyceraldehyde-3-phosphate dehydrogenase activity in inhibited.

The binding of NADH and NADPH to bovine-liver glutamate dehydrogenase. Spectroscopic characterisation.

1. The binding of NADH and NADPH to bovine liver glutamate dehydrogenase has been studied using circular dichroism (CD) and fluorescence spectroscopy, under comparable conditions of enzyme concentration. Spectroscopic titrations have been analysed using non-linear least-squares techniques to determine dissociation constants and parameters for the different chromophoric portions of the bound coenzymes, according to different models for the binding equilibria. 2. For NADPH, enhancement of dihydronicotinamide fluorescence and positive CD at 340 nm reflect binding of the coenzyme at the active site (site I) via the dihydronicotinamide moiety with a dissociation constant K1 of 50 microM; negative CD at 260 nm reflects the binding of a second molecule of coenzyme at the non-active site (site II) via the adenine moiety with a KII of 650 microM. 3. For NADH, the same spectroscopic assignment is made; however, site II affinity is much greater (KII 20 microM) and exceeds the affinity at the active site (KI 50 microM). 4. In no case was spectroscopic evidence obtained with either coenzyme to support the concept of non-identical active site binding as three sites IA and three sites IB per hexamer. 5. Coenzyme affinities are approximately twofold higher in triethanolamine buffer (0.05 + 0.1 M KCl, pH 7.6) compared with phosphate buffer (0.1 M at pH 7.0). 6. ADP enhances the CD of bound NADPH at 340 nm by up to twofold; phosphate ion also enhances the CD and binds with a Kd of 60-80 mM. GTP causes strong negative CD at 340 nm but only for the NADPH bound at site II. These results are consistent with independent binding sites for ADP (site II) and GTP (site III); phosphate ion is apparently able to affect certain properties of all three sites.

Subunit dissociation and unfolding of bovine liver glutamate dehydrogenase induced by guanidine hydrochloride

Equilibrium and kinetic measurements were carried out on the denaturation of bovine liver glutamate dehydrogenase ( l-glutamate:NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3)induced by guanidine hydrochloride (Gdn-HCl) in 0.2 M phosphate buffer (pH 7.3) using two types of detection, light-scattering and circular dichroism. The results obtained in equilibrium studies showed that the enzyme exists in solution as hexamers of native subunit at Gdn-HCl concentrations below 0.6 M, as trimers of native subunit in the concentration range between 1.0 and 2.0 M, and as monomers with unfolded structure above 2.8 M. From the kinetic studies, it was found that the dissociation of hexamer to trimer takes place more rapidly than that of trimer to monomer by a factor of 10, and it was also found that the unfolding of the polypeptide chain occurs much more slowly than subunit dissociation.

Theory and applications of the transferred nuclear overhauser effect to the study of the conformations of small ligands bound to proteins

The principles, theory, and applications of the transferred proton-proton nuclear Overhauser effect (TRNOE) to the study of the conformations of small molecules to proteins are presented and discussed. The basis of the TRNOE involves the transfer of information concerning cross relaxation between two bound ligand nuclei from the bound to the free state by chemical exchange so that negative NOEs on the easily detectable free or observed ligand resonances may be seen following irradiation of other ligand resonances (free, bound, or observed), thus conveying information on the proximity in space of bound ligand nuclei. In the presence of protein, a negative TRNOE on either the free or observed resonance of nucleus i will be observed following irradiation of either the free, bound, or observed resonance of nucleus j, providing several conditions are met. Methods for obtaining quantitative conformational information from TRNOE measurements are discussed. The TRNOE method is applicable even when no individual proton resonances of either the protein or the bound ligand can be resolved, and is not limited by the molecular weight of the protein, extending the molecular weight range over which 1H NMR can provide useful conformational information to the very largest systems. This is illustrated by the determination of the glycosidic bond torsion angle of adenosine 5′-monophosphate bound to horse liver alcohol dehydrogenase, yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase.

Regulation of bovine glutamate dehydrogenase. The effects of pH and ADP.

The activity of bovine liver glutamate dehydrogenase is affected in several ways depending on substrate concentrations and pH. At ph 6.5 and below, both oxidative deamination and reductive amination reactions are inhibited by ADP. At pH 7.0 and above both activatory and inhibitory effects can be observed depending on substrate concentrations. The effects are explicable in terms of a model with ADP binding at both a regulatory site and competing with coenzyme at the active site. The activatory effects of ADP result from destabilization of various abortive complexes by ADP binding to its regulatory site. The concerted effects of pH and ADP lead to a potentiation of either activation effects or inhibition effects depending on conditions. A consideration of in vivo concentrations of the various substrates involved and intramitochondrial pH and adenine nucleotide levels suggests that in vivo the reductive amination reaction is favored. It is suggested that glutamate dehydrogenase may be intimately involved with regulation of the urea cycle by responding to changes in the mitochondrial ammonia levels.

Alpha-Ketoglutaric acid: solution structure and the active form for reductive amination by bovine liver glutamate dehydrogenase.

A study of the various solution forms of alpha-ketoglutaric acid using UV absorption spectrophotometry and 13C NMR spectroscopy shows that at neutral pH alpha-ketoglutarate exists predominantly as the keto form with about 7% hydrated form (gem-diol) and a small amount of cyclic form. Protonation of the gamma-carboxylate group increases the amount of cyclic form to 20% with very little increase in the amount of gem-diol. Protonation of the alpha-carboxylate increases the amount of cyclic form to 30% and the amount of gem-diol to 35%. The pH and temperature dependence of the 13C NMR line widths indicated that the interconversion of keto and cyclic forms is extremely rapid. The rate of interconversion of keto and gem-diol forms was studied spectrophotometrically at various temperatures, buffers, and pH values and by varying the total concentration of alpha-ketoglutarate. The hydration reaction of alpha-ketoglutarate is not catalyzed by bovine liver glutamate dehydrogenase (E) or by the E-NADPH complex. The enzyme uses the keto form of alpha-ketoglutarate as the substrate. The gem-diol form is not itself a substrate but becomes converted to the keto form with a half-life of about 0.3 min at 15 degrees C in 0.1 M potassium phosphate buffer (pH 7.6).

Kinetic studies to determine the mechanism of regulation of bovine liver glutamate dehydrogenase by nucleotide effectors.

A combination of kinetic and isotope effect studies in the presence and absence of the effectors ADP and GTP was used to elucidate the mechanism of regulation of bovine liver glutamate dehydrogenase. ADP at low concentrations of glutamate competes with TPN for free enzyme. GTP exhibits a similar effect at high concentrations (100 microM and above). When ADP binds at its allosteric site, it increases the off rates of both alpha-ketoglutarate and TPNH from their product complexes. This results in a decrease in V/K for both substrates, an increase in V, and an increase in the deuterium isotope effects for all three parameters so that they are all about 1.3. The rate of release of glutamate from E-TPNH-glutamate is also apparently enhanced since no substrate inhibition by glutamate is observed in the presence of ADP. The effect of GTP is in opposition to that of ADP in that GTP decreases the off rates for both TPN and glutamate from E-TPN-glutamate as well as the off rates for alpha-ketoglutarate and TPNH. This results in an increase in the V/K's for both substrates, a decrease in V, and a decrease in the deuterium isotope effects for all three parameters to a value of 1. Substrate inhibition by glutamate is also eliminated by GTP probably by preventing any significant accumulation of E-TPNH to which glutamate binds as an inhibitor.

Regulation of coenzyme utilization by bovine liver glutamate dehydrogenase: Investigations using thionicotinamide analogues of NAD and NADP in a dual wavelength assay

1. 1. The coenzyme preference of bovine liver glutamate dehydrogenase (GDH) was probed using dual wavelength spectroscopy and pairing the thionicotinamide analogues, S-NAD or S-NADP (which have absorbance maxima at 400 nm), with the natural coenzymes, NADP or NAD. 2. 2. S-NAD and S-NADP were found to be good alternate substrates for GDH : the apparent Kin m's for the thioderivatives were similar to those of the corresponding natural coenzymes, the apparent Kin m's for glutamate were unaltered by the substitution of the thioderivatives, and the effects of inhibitors and activators on S-NAD or S-NADP kinetics were qualitatively the same as those found for NAD or NADP, respectively. 3. 3. Dual wavelength assays paired NAD and S-NADP or S-NAD and NADP to study the simultaneous reduction of the two coenzymes. Conditions of increasing glutamate concentrations produced differential effects on the rates of the NAD vs NADP reactions, the result, with either nucleotide pair, promoting the NADP linked reaction. 4. 4. Activators and inhibitors of the GDH reaction also showed differential effects upon the NAD vs NADP linked reaction rates in the dual wavelength assay. ADP and leucine, which activate both the NAD and the NADP linked reactions in single coenzyme assays, preferentially activate the NADP or S-NADP linked reactions in the dual nucleotide assays. GTP produced greater inhibition of the NAD or S-NAD linked reactions than of the NADP or S-NADP reactions while ATP inhibited NAD or S-NAD reactions and activated NADP or S-NADP reactions. The net effect of all metabolite modulators was to promote the NADP linked reaction by decreasing the activity ratios, ν (Nad)/ν (S-Nadp) or ν (S-Nad)/ν (Nadp). 5. 5. The results are consistent with the suggestion that NADP is the preferred coenzyme for the oxidative deamination of glutamate by GDH even though the enzyme is capable of utilizing either coenzyme in vitro.

Effect of phosphate concentration on the kinetics of bovine liver glutamate dehydrogenase self-association

The kinetics of the self-association of bovine liver glutamate dehydrogenase was studied at various phosphate buffer concentrations (pH 7.3) at 11.5°C by means of the temperature-jump technique with scattered light detection. The observed relaxation times were well explained by the random association model of Thusius et al. With increasing phosphate concentration, the association rate constant derived from the model decreased, while the dissociation rate constant was left almost constant. Relaxation amplitude was also dependent on the phosphate concentration. The changes in the rate constant and relaxation amplitude with phosphate concentration are well elucidated by assuming that glutamate dehydrogenase is protected from association by specific masking of the association site by phosphate ions.