The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides

1. The binding of NAD(+) and NADP(+) to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD(+) the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD(+) and NADP(+), in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD(+) or NADP(+), the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.     

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 reaction of bovine glutamate dehydrogenase with periodate-oxidised ADP

1. The reactive analogue oADP produced by periodate oxidation of ADP has been studied as a potential affinity label for the enzyme bovine glutamate dehydrogenase, using circular dichroism (CD) difference spectroscopy to monitor specific binding. 2. The analogue binds stoichiometrically, rapidly and reversibly to the adenine nucleotide binding site with Kd approximately equal to 12 microM (20 degrees C, pH 7) with characteristic intensification of the adenine nucleotide CD at 260 nm. 3. This complex is unstable and decays with a half-life of about 1.5 h; the analogue then becomes attached as a Schiff base to a number of subsidiary sites, including the enzyme active site, with partial inactivation of the enzyme. 4. Depending upon initial concentration of oADP, the enzyme activity is progressively lost during the slow reaction; following borohydride reduction, up to four molecules of analogue are bound/subunit. 5. Protection against loss of enzyme activity is afforded by the coenzyme NAD+ plus glutarate or L-hydroxyglutarate (an effective inhibitor), or by glutarate alone, but not by NAD+ alone. 6. Spectroscopic and protection studies indicate that after the decay of the specific CD signal, the enzyme retains the capacity to bind ADP, but that this is progressively lost in parallel with decay of enzymic activity. 7. The results are consistent with proximity or functional interaction between the adenine nucleotide site and the coenzyme binding portion of the active site. 8. Thus oADP does not act as a true affinity label for the adenine nucleotide binding site, but the reaction subsequent to binding at that site shows some specificity directed towards the active site.     

Thermodynamic resolution of cytochrome P450 and characterization of an iron-sulfur center in rat liver microsomal preparations.

Binding of 8-anilinonaphthalene sulfonate (ANS) to glutamate dehydrogenase results in enzyme inhibition and a marked increase in the fluorescence of ANS. Perphenazine and GTP increase the fluorescence of ANS-glutamate dehydrogenase secondary to their known ability to alter the conformation of this enzyme. Aspartate aminotransferases, which form enzyme-enzyme complexes with glutamate dehydrogenase, produce a slight decrease in the fluorescence of ANS-glutamate dehydrogenase.While ANS and perphenazine are allosteric inhibitors of reactions catalyzed by free glutamate dehydrogenase, they do not inhibit reactions catalyzed by aminotransferaseglutamate dehydrogenase complexes. This is in spite of the fact that the aminotransferase does not prevent either ANS or perphenazine from being bound to glutamate dehydrogenase. Therefore, reactions catalyzed by the enzyme-enzyme complex are apparently not inhibited by ANS or perphenazine because binding of the aminotransferase to glutamate dehydrogenase prevents these ligands from altering the conformation of glutamate dehydrogenase. This is consistent with the fact that the aminotransferase also prevents perphenazine from enhancing the fluorescence of ANS-glutamate dehydrogenase.Reactions catalyzed by the enzyme-enzyme complex are inhibited by GTP and the aminotransferase does not prevent GTP from enhancing the fluorescence of ANS-glutamate dehydrogenase. Therefore, binding of the aminotransferase to glutamate dehydrogenase does not prevent GTP from altering the conformation of glutamate dehydrogenase.The fact that the aminotransferase completely prevents perphenazine from increasing the fluorescence of ANS-glutamate dehydrogenase suggests that in the enzymeenzyme complex each glutamate dehydrogenase polypeptide chain can be bound to an aminotransferase polypeptide chain. This would mean that three aminotransferase molecules can be bound to each monomeric unit (M_r 3 × 10⁵) of glutamate dehydrogenase.     

Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

Formation of transient complexes in the glutamate dehydrogenase catalyzed reaction.

The reaction of glutamate dehydrogenase and glutamate (gl) with NAD+ and NADP+ has been studied with stopped-flow techniques. The enzyme was in all experiments present in excess of the coenzyme. The results indicate that the ternary complex (E-NAD(P)H-kg) is present as an intermediate in the formation of the stable complex (E-NAD(P)H-gl). The identification of the complexes is based on their absorption spectra. The binding of the coenzyme to (E-gl) is the rate-limiting step in the formation of (E-NAD(P)H-kg) while the dissociation of alpha-ketoglutarate (kg) from this complex is the rate-limiting step in the formation of (E-NAD(P)H-gl). The Km for glutamate was 20-25 mM in the first reaction and 3 mM in the formation of the stable complex. The Km values were independent of the coenzyme. The reaction rates with NAD+ were approximately 50% greater than those with NADP+. Furthermore, high glutamate concentration inhibited the formation of (E-NADH-kg) while no substrate inhibition was found with NADP+ as coenzyme. ADP enhanced while GTP reduced the rate of (E-NAD(P)H-gl) formation. The rate of formation of (E-NAD(P)H-kg) was inhibited by ADP, while it increased at high glutamate concentration when small amounts of GTP were added. The results show that the higher activity found with NAD+ compared to NADP+ under steady-state assay conditions do not necessarily involve binding of NAD+ to the ADP activating site of the enzyme. Moreover, the substrate inhibition found at high glutamate concentration under steady-state assay condition is not due to the formation of (E-NAD(P)H-gl) as this complex is formed with Km of 3 mM glutamate, and the substrate inhibition is only significant at 20-30 times this concentration.     

A re-examination of the reaction between ox liver glutamate dehydrogenase and 1-fluoro-2,4-dinitrobenzene.

Reaction of ox liver glutamate dehydrogenase with 1-fluoro-2,4-dinitrobenzene for 4 h at pH 8 caused 86% inactivation, almost complete desensitization to allosteric inhibition by GTP, but only partial desensitization to ADP activation. The enzyme remained hexameric after such treatment. NAD⁺, but not NADH or NADPH, partially protected activity. Protection was enhanced by GTP and decreased by ADP. GTP and NADH together protected effectively, although separately neither protected. GTP and NADPH gave partial protection of activity. Glutarate and succinate, inhibitors competitive with glutamate, gave substantial protection, slightly enhanced in the presence of NAD⁺. With glutarate, but not succinate, an initial activation was seen during chemical modification. The allosteric response to GTP was protected by GTP itself only when NAD⁺ or NAD(P)H was also present; other ligands failed to protect. Similarly ADP alone did not protect ADP sensitivity. NADH partially protected ADP sensitivity, although NADPH did not. ADP itself counteracted the protection given by NADH. GTP with NADH completely protected ADP sensitivity. This combination of ligands thus protects all the assayed properties. GTP with NADPH gave less complete protection of the ADP response. Observed protection patterns varied with the pH and coenzyme concentration of the assay mixture under constant conditions of chemical modification. Overall, the results are inconsistent with the view that dinitrophenylation directly blocks nucleotide binding sites, and suggest rather that it interferes with communication between sites.     

Protection of glutamate dehydrogenase by nicotinamide–adenine dinucleotide against reversible inactivation by pyridoxal 5′-phosphate as a sensitive indicator of conformational change induced by substrates and substrate analogues

1. The steady residual activity of ox liver glutamate dehydrogenase at equilibrium with the reversible inactivator pyridoxal 5'-phosphate was measured in the presence and absence of various protecting agents. 2. NAD(+) (up to 15mm) and its 3-acetylpyridine analogue (up to 5mm) both failed to protect, in contrast with NADH. 3. Partial protection was given by glutarate and by succinate. Adipate and pentanoate were much less effective. 4. Correspondingly, whereas succinate and glutarate were both shown to be strong inhibitors of the catalytic reaction, competitive with glutamate, adipate was only weakly competitive, and the still weaker inhibition by pentanoate was non-competitive. 5. When the enzyme was saturated with glutarate, NAD(+) became a good, although still partial, protecting agent. In the absence of protection, 1.8mm-pyridoxal 5'-phosphate decreased enzyme activity to 9%, in the presence of 150mm-glutarate to 29%, and with glutarate and 1mm-NAD(+) only to 73%. 6. 2-Oxoglutarate also promoted protection by NAD(+), but neither pentanoate nor succinate did so. The finding with succinate is remarkable in view of findings 3 and 4 above. 7. It seems possible that substrates or analogues possessing the glutarate structure promote a conformational change that alters the mode of NAD(+) binding. This may explain why glutamate is a much better substrate than norvaline or aspartate and why negative interactions in coenzyme binding occur only in the formation of ternary complexes with glutamate or its analogues.     

Adenosine 5'-0-[S-(4-succinimidyl-benzophenone)thiophosphate]: a new photoaffinity label of the allosteric ADP site of bovine liver glutamate dehydrogenase

By reaction of adenosine 5'-monothiophosphate with benzophenone-4-maleimide, we synthesized adenosine 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (AMPS-Succ-BP) as a photoreactive ADP analogue. Bovine liver glutamate dehydrogenase is known to be allosterically activated by ADP, but the ADP site has not been located in the crystal structure of the hexameric enzyme [Peterson, P. E., and Smith, T. J. (1999) Structure 7, 769-782]. In the dark, AMPS-Succ-BP reversibly activates GDH. Irradiation of the complex of glutamate dehydrogenase and AMPS-Succ-BP at lambda >300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The k(obs) for photoactivation shows nonlinear dependence on the concentration of AMPS-Succ-BP, with K(R) = 4.9 microM and k(max) = 0.076 min(-)(1). The k(obs) for photoreaction by 20 microM AMPS-Succ-BP is decreased 10-fold by 200 microM ADP, but is reduced less than 2-fold by NAD, NADH, GTP, or alpha-ketoglutarate. Modified enzyme is no longer activated by ADP, but is still inhibited by GTP and high concentrations of NADH. These results indicate that reaction of AMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [(3)H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys(488)-Glu(495) has been identified as the only reaction target, and the data suggest that Arg(491) is the modified amino acid. Arg(491) (in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or near the enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned within the crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of the enzyme.     

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe

Time-resolved fluorescence spectroscopy was used to investigate the solution dynamics of Escherichia coli tRNAPhe, Phe-tRNAPhe, and Phe-tRNAPhe associated with GTP and elongation factor Tu (EF-Tu) in a ternary complex. Two fluorescence probes were employed: fluorescein, covalently bound to Phe-tRNAPhe at the s4U8 base (Phe-tRNAPhe-Fl8), and ethidium bromide, noncovalently associated with the tRNA (EB.Phe-tRNAPhe). The lifetimes observed for ethidium bromide were 1.89 ns, free in solution, and 26.3 ns, bound to its tight binding site on tRNA. Fluorescein-labeled tRNA had a lifetime of 4.3 ns, with no significant difference among the values for aminoacylated, unacylated, and EF-Tu-bound Phe-tRNAPhe-Fl8. Differential phase and modulation data for each fluorophore-tRNA system were fit with local and global Debye rotational relaxation times. Local motion of the labeled fluorescein in Phe-tRNAPhe-Fl8, tRNAPhe-Fl8, and Phe-tRNAPhe-Fl8.EF-Tu.GTP was characterized by rotational relaxation times of 2.7 +/- 0.5, 2.4 +/- 0.4, and 2.4 +/- 0.1 ns, respectively. These values are equal, within experimental error, and suggest that the rotational mobility of the s4U8-conjugated dye is unaffected by either tRNAPhe aminoacylation or ternary complex formation. Global rotational relaxation times for Phe-tRNAPhe-Fl8, 97 ns, and EB.Phe-tRNAPhe, 140 ns, were equivalent to those determined for the unacylated species, denoting little change in the overall size or shape of the tRNA molecule upon aminoacylation. These values for (Phe-)tRNA were larger than expected for a hydrated sphere of equivalent volume, 83 ns, and therefore confirm the asymmetric nature of the tRNA structure in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation

Glutamate dehydrogenase is found in all organisms and catalyses the oxidative deamination of l-glutamate to 2-oxoglutarate. However, only animal GDH utilizes both NAD(H) or NADP(H) with comparable efficacy and exhibits a complex pattern of allosteric inhibition by a wide variety of small molecules. The major allosteric inhibitors are GTP and NADH and the two main allosteric activators are ADP and NAD(+). The structures presented here have refined and modified the previous structural model of allosteric regulation inferred from the original boGDH.NADH.GLU.GTP complex. The boGDH.NAD(+).alpha-KG complex structure clearly demonstrates that the second coenzyme-binding site lies directly under the "pivot helix" of the NAD(+) binding domain. In this complex, phosphates are observed to occupy the inhibitory GTP site and may be responsible for the previously observed structural stabilization by polyanions. The boGDH.NADPH.GLU.GTP complex shows the location of the additional phosphate on the active site coenzyme molecule and the GTP molecule bound to the GTP inhibitory site. As expected, since NADPH does not bind well to the second coenzyme site, no evidence of a bound molecule is observed at the second coenzyme site under the pivot helix. Therefore, these results suggest that the inhibitory GTP site is as previously identified. However, ADP, NAD(+), and NADH all bind under the pivot helix, but a second GTP molecule does not. Kinetic analysis of a hyperinsulinism/hyperammonemia mutant strongly suggests that ATP can inhibit the reaction by binding to the GTP site. Finally, the fact that NADH, NAD(+), and ADP all bind to the same site requires a re-analysis of the previous models for NADH inhibition.     

Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes.

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.     

The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.     

Effect of pH and ionic strength on the catalytic and allosteric properties of native and chemically modified ox liver mitochondrial glutamate dehydrogenase.

Inorganic phosphate, a strong activator of glutamate dehydrogenase at pH 8.0–9.0, is an inhibitor at pH 6.0–7.6. The extent of inhibition increases with the decrease of pH. The same effect is shown by other electrolytes, including Tris-hydroxymethyl-aminomethane and NaCl.The combined effect of pH and ionic strength also alters the allosteric characteristics of the enzyme. Lowering the pH minimizes the activation by high concentrations of NAD; phosphate partially restores this activation. The allosteric activation by ADP disappears at pH around neutrality; in the pH range 6.0–7.0, ADP becomes a strong inhibitor, the inhibition being enhanced by the addition of ionic compounds. Similarly, the extent of allosteric inhibition by guanosine 5′-triphosphate (pyro) (GTP), which is maximal at pH 9.0, decreases at lower pH values and a slight activation is observed in the presence of electrolytes at pH 6.0.Glutamate dehydrogenase, selectively desensitized by dinitrophenylation in the presence of ADP, can be activated by ADP at pH 9.0, but is no longer inhibited by the same effector at pH 6.0, high salt concentration. The densensitized enzyme is not inhibited by GTP at pH 9.0, but is activated by this effector at pH 6.0 in the presence of ionic compounds. Conversely, GTP-protected dinitrophenylated glutamate dehydrogenase is desensitized only to the effect of the activating modifier, ADP at pH 9.0, GTP at pH 6.0, high salt concentration. These findings suggest that the conformation of each allosteric site of glutamate dehydrogenase is changed by pH and ionic strength so that it keeps its specificity for the ligand which brings about a given effect, activation or inhibition, independently from its chemical structure.     

The reaction of a histidine residue in glutamate dehydrogenase with diethyl pyrocarbonate

1. One mol of diethyl pyrocarbonate will react with one mol of glutamate dehydrogenase polypeptide chains to form one mol of N(1)-carbethoxyhistidine. Reaction is prevented by NADH. 2. The 1:1 complex has an increased specific activity (1.4-2.0-fold). 3. The reason for the activation is discussed. The results are not consistent with NADH dissociation from the enzyme-glutamate-NADH complex being rate-limiting in the steady state measured. 4. The effects of modification on the properties of the enzyme were investigated. The effects of GTP and NAD(+) on the enzyme activity are unaltered by activation. NADH binding is unaltered and there is no apparent change in the molecular weight. However, the activated enzyme can still be further activated by ADP. K(s) for ADP is decreased fivefold.     

Regulatory effects of purine nucleotide analogs with liver glutamate dehydrogenase.

A total of 26 different purine nucleotides with specific modifications in the base moiety and/or in the polyphosphate chain as well as various combinations of nucleotides were tested as allosteric effectors of beef liver glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3). The capacity of these nucleotide analogs to activate or to inhibit the glutamate dehydrogenase activity is expressed quantitatively and scaled between the extreme effects of ADP and GTP, respectively. The significance of distinct structural elements for the enzyme-effector interaction is discussed. While the inhibitory GTP site is less specific, accepting many natural and most modified nucleoside triphosphates as inhibitors, the activating ADP site shows a much higher specificity for nucleotides as activators.     

Use of a fluorescent probe for the study of the active center of D-glyceraldehyde-3-phosphate dehydrogenase]

The effect of NAD on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to yeast glyceraldehyde-3-phosphate dehydrogenase has been studied using difference spectrophotometric and fluorescence techniques. Coenzyme addition causes the displacement of ANS from its complex with the dehydrogenase, as suggested by the effect of NAD on the fluorescence of the enzyme--ANS complex, as well as on the magnitude of the difference spectrum of the complex. Adenine containing NAD fragments, adenosine, 5'-AMP, and ADP were shown to compete with ANS for the common site on the enzyme using fluorimetric technique; in the case of adenosine and 5'-AMP a direct method of analytical ultracentrifugation was also employed. The results obtained by both methods suggest the dye binding at the adenine subsite of the dehydrogenase. The interaction with ANS causes no detectable conformational changes of the protein. The fluorescence of the dye-enzyme complex increases and the emission maximum shifts to shorter wavelengths on addition of nicotinamide mononucleotide. This suggest some conformational changes to occur in the microenvironment of the bound dye in response to the interaction with the ligand in the nicotinamide subsite. The participation of the nicotinamide subsite of the active center in determining the character of conformational transitions associated with coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase is discussed.     

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.     

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%.     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.