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.     

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.     

Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli

Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.     

Thermodynamics of protein-peptide interactions in the ribonuclease-S system studied by molecular dynamics and free energy calculations.

Hydrophobic interactions between the S-peptide and S-protein in the ribonuclease-S complex are probed using molecular dynamics simulations and free energy calculations. Three successive mutations at the buried position Met13 are simulated: Met----Leu, Leu----Ile, and Ile----Val, for which X-ray structures and experimental thermodynamic data are available. The calculations give theoretical estimates of the changes in binding free energies associated with these mutations. The calculated free energy differences are small (0-1.6 kcal/mol), in agreement with experiment. However the simulated structures deviate significantly from the experimental ones (mean deviation approximately 1.5-2 A), and a large uncertainty in the calculated free energies (1-2 kcal/mol) arises from the multiple minimum problem. Indeed, multiple conformations are available to the side chains around the mutation site, and the sampling of dihedral rotamer transitions is limited, despite long simulations. Fluctuations within each local minimum give rise to a small statistical error. However the uncertainty due to multiple conformations is much greater than the uncertainty due to random statistical errors. In our work, an artificial cancellation of errors arose because we studied conformations of the RNase complex and of the S-peptide that were very similar. In general, the criterion for a precise simulation is not merely to reduce the random statistical error, as has been suggested, but rather to sample all the important local minima along the mutation pathway, and to reduce the statistical error for each one. Our calculations suggest that the packing changes associated with the mutations are energetically small and localized, and largely cancel when the complex and the S-peptide are compared. Solvation of the methionine side chain partial charges in the S-peptide and the complex appear to be energetically equivalent, so that removing them (as in Met13----Leu, Ile, Val) does not affect binding. Enthalpy and entropy changes could not be estimated reliably.     

The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery.

BACKGROUND: Bovine glutamate dehydrogenase (boGDH) is a homohexameric, mitochondrial enzyme that reversibly catalyzes the oxidative deamination of L-glutamate to 2-oxoglutarate using either NADP(H) or NAD(H) with comparable efficacy. GDH represents a key enzymatic link between catabolic and biosynthetic pathways, and is therefore ubiquitous in both higher and lower organisms. Only mammalian GDH exhibits strong negative cooperativity with respect to the coenzyme, however, and is regulated by a large number of allosteric effectors. RESULTS: The atomic structure of boGDH in complex with NADH, glutamate, and the allosteric inhibitor GTP has been determined to 2.8 A resolution. The major difference between the bacterial and bovine GDH structures is the presence of an additional 'antenna' in boGDH that protrudes from each trimer, twisting counterclockwise along the threefold axis. NADH and glutamate are clearly observed in the active site, but the contacts differ slightly from those observed in Clostridium symbiosum GDH. A second, inhibitory NADH molecule lies buried in the core of the hexamer. Finally, two GTP molecules bind near the hinge region connecting the NAD(+)- and glutamate-binding domains. CONCLUSIONS: We propose that the antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation. GTP and NADH inhibit GDH by keeping the catalytic cleft in a closed conformation. In contrast, ADP probably binds to the back of the NAD(+)-binding domain and activates the enzyme by keeping the catalytic cleft open. Extensive contacts between antennae within the crystal lattice may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix.     

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.     

Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation

Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Unlike GDH from bacteria, mammalian GDH exhibits negative cooperativity with respect to coenzyme, activation by ADP, and inhibition by GTP. Presented here are the structures of apo bovine GDH, bovine GDH complexed with ADP, and the R463A mutant form of human GDH (huGDH) that is insensitive to ADP activation. In the absence of active site ligands, the catalytic cleft is in the open conformation, and the hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals. This is consistent with the fact that ADP promotes aggregation in solution. ADP is shown to bind to the second, inhibitory, NADH site yet causes activation. The beta-phosphates of the bound ADP interact with R459 (R463 in huGDH) on the pivot helix. The structure of the ADP-resistant, R463A mutant of human GDH is identical to native GDH with the exception of the truncated side chain on the pivot helix. Together, these results strongly suggest that ADP activates by facilitating the opening of the catalytic cleft. From alignment of GDH from various sources, it is likely that the antenna evolved in the protista prior to the formation of purine regulatory sites. This suggests that there was some selective advantage of the antenna itself and that animals evolved new functions for GDH through the addition of allosteric regulation.     

Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase

The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked inhibition.     

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.     

Allosteric nucleotide specificity of phosphorylase kinase: correlation of binding, conformational transitions, and activation. Utilization of lin-benzo-ADP to measure the binding of other nucleoside diphosphates, including the phosphorothioates of ADP

Recent work has shown that ADP is an allosteric activator of nonphosphorylated phosphorylase kinase from rabbit skeletal muscle (Cheng, A., Fitzgerald, T. J., and Carlson, G. M. (1985) J. Biol. Chem. 260, 2535-2542). The specificity of the allosteric site for nucleoside diphosphates is further investigated in this study. Only purine nucleoside diphosphates are capable of causing allosteric activation, and an amino group at position 2 or 6 of the purine ring is required. Comparisons are made of the abilities of 5'-diphosphate analogs of ADP, including phosphorothioates, to activate, to bind, and to induce in the enzyme's beta subunits conformational changes associated with activation. Binding is measured by competition titrations utilizing fluorescence polarization of lin-benzo-ADP, itself an allosteric activator; and conformational changes are measured by partial proteolysis and chemical cross-linking. When measured at an identical percentage of saturation at the allosteric site, the abilities of ADP analogs to induce conformational changes in the beta subunits parallel their abilities to activate the holoenzyme. An unmodified beta-phosphate of ADP, although not necessary for binding at the allosteric site, is needed to fully drive the activating conformational transition. The activating nucleoside diphosphate appears to be the free species, as opposed to its Mg2+ complex.     

The Fe-CO bond energy in myoglobin: a QM/MM study of the effect of tertiary structure

The Fe-CO bond dissociation energy (BDE) in myoglobin (Mb) has been calculated with B3LYP quantum mechanics/molecular mechanics methods for 22 different Mb conformations, generated from molecular dynamics simulations. Our average BDE of 8.1 kcal/mol agrees well with experiment and shows that Mb weakens the Fe-CO bond by 5.8 kcal/mol; the calculations provide detailed atomistic insight into the origin of this effect. BDEs for Mb conformations with the R carbonmonoxy tertiary structure are on average 2.6 kcal/mol larger than those with the T deoxy tertiary structure, suggesting two functionally distinct allosteric states. This allostery is partly explained by the reduction in distal cavity steric crowding as Mb moves from its T to R tertiary structure.     

Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase

The adenine binding domain of the ADP site within human glutamate dehydrogenase (GDH) was identified by cassette mutagenesis at the Tyr187 position. The wild type GDH was activated 3-fold by ADP at a concentration of 1 mM at pH 8.0, whereas no significant activation by ADP was observed with the Tyr187 mutant GDH regardless of the size, hydrophobicity, and ionization of the side chains. Studies of the steady-state velocity of the mutant enzymes revealed essentially unchanged apparent K(m) values for 2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective apparent V(max) values. The binding of ADP to the wild type or mutant GDH was further examined by photoaffinity labeling with [alpha-(32)P]8-azidoadenosine 5'-diphosphate (8N(3)ADP). 8N(3)ADP, without photolysis, mimicked the stimulatory properties of ADP on GDH activity. Saturation of photoinsertion with 8N(3)ADP occurred with apparent K(d) values near 25 microM for the wild type GDH, and the photoinsertion of [alpha-(32)P]8N(3)ADP was decreased best by ADP in comparison to other nucleotides. Unlike the wild type GDH, essentially no photoinsertion was detected for the Tyr187 mutant GDH in the presence or absence of 1 mM ADP. For the wild type GDH, photolabel-containing peptide generated by tryptic digestion was identified in the region containing the sequence EMSWIADTYASTIG, and the photolabeling of this peptide was prevented >95% by the presence of 1 mM ADP during photolysis, whereas no such a peptide was detected for the Tyr187 mutant GDH in the presence or absence of ADP. These results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide is within the ADP binding domain of the human GDH and that Tyr187 is responsible for the efficient base binding of ADP to human GDH.     

Conformational flexibility of a model protein upon immobilization on self-assembled monolayers

The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.     

Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.     

Free energy perturbation calculations on binding and catalysis after mutating threonine 220 in subtilisin

We present the results of free energy perturbation calculations on binding and catalysis of a tetrapeptide substrate, acetyl-Phe-Ala-Ala-Phe-NMe, by native subtilisin BPN' and a subtilisin BPN' mutant (Thr220----Ala220). The calculated difference in the free energy of binding was 0.70 +/- 0.72 kcal/mol. The calculated difference in the free energy of catalysis was 1.48 +/- 0.89 kcal/mol. These calculated values compare well with the experimental values in which another substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, was used. These findings suggest that Thr220 is more important for catalysis than substrate binding.     

Comparison of AMP and NADH binding to glycogen phosphorylase b

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.     

Calculation of cyclodextrin binding affinities: energy, entropy, and implications for drug design

The second generation Mining Minima method yields binding affinities accurate to within 0.8 kcal/mol for the associations of alpha-, beta-, and gamma-cyclodextrin with benzene, resorcinol, flurbiprofen, naproxen, and nabumetone. These calculations require hours to a day on a commodity computer. The calculations also indicate that the changes in configurational entropy upon binding oppose association by as much as 24 kcal/mol and result primarily from a narrowing of energy wells in the bound versus the free state, rather than from a drop in the number of distinct low-energy conformations on binding. Also, the configurational entropy is found to vary substantially among the bound conformations of a given cyclodextrin-guest complex. This result suggests that the configurational entropy must be accounted for to reliably rank docked conformations in both host-guest and ligand-protein complexes. In close analogy with the common experimental observation of entropy-enthalpy compensation, the computed entropy changes show a near-linear relationship with the changes in mean potential plus solvation energy.     

Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain

Glutamate dehydrogenase (GDH) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low-speed pellet (480 g for 10 min). Approximately 60% of the high-speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates alpha-ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L-leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 microM GTP for the soluble and particulate fractions, respectively. The Hill plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45 degrees C and pH 7.4 in 50 mM phosphate buffer.     

Energy of binding of Aspergillus oryzae beta-glucosidase with the substrate, and the mechanism of its enzymic action

Several beta-D-glucopyranosides (p-nitrophenyl, phenyl, and ethyl), 1-thio-beta-D-glucopyranosides, and phenyl 2-deoxy, 3-deoxy, 4-deoxy, and 6-deoxy beta-D-glucopyranosides were synthesized and used to study the mechanism of the enzymatic action of Taka-beta-glucosidase [EC 3.2.1.21 Aspergillus oryzae]. Kinetic constants of the enzyme for these glycosides were determined from S/V-S or 1/V-1/S plots, and the hydrolysis rates of these compounds with the enzyme, acid (3 N HCl) and alkali (3 N NaOH) were compared. Inhibition of the enzyme by 1,5-anhydroglucitol, glucal, dihydroglucal, and 1,6-anhydroglucopyranose was also examined. Glucal and 1,5-anhydroglucitol showed strong competitive inhibition. Free energy of binding of each hydroxyl group of glucosidic glucose with the enzyme was estimated from Kms of phenyl beta-glucoside and its deoxy analogues, and also Ki values of some inhibitors. The free energies of binding of 2-OH, 3-OH, 4-OH, and 6-OH were calculated to be 1.1, 2.4, 0.7, and 1.8 kcal/mol, respectively. The free energy of binding of phenoxide at C-1 (0.3 kcal/mol) was calculated from the Km of Ph-beta-Glc and Ki of 1,5-anhydroglucitol. The energy of binding of 5-CH2OH (2.3 kcal/mol) was obtained from the Km of Ph-beta-Glc and that of Ph-beta-Xyl. The sum (6.8 kcal/mol) of each partial binding free energy was close to the value of binding free energy of Ph-beta-Glc (7.0 kcal/mol) calculated by the equation; -delta Gbind = -RT ln Km-T delta Smix, showing that the methods of estimation of each binding energy used in the present study seemed reasonable. Glucal, having a pyranose form distorted slightly, showed strong competitive inhibition and the Ki of this inhibitor was smaller than the Km of Ph-beta-Glc, suggesting that the sugar ring bound to the active site was distored to a half chair form which is labile to acid hydrolysis.