Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.     

The structure and allosteric regulation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a ‘housekeeping’ enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an ‘antenna’ like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.     

The structure and allosteric regulation of mammalian glutamate dehydrogenase

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.     

Structural requirements for the binding of AMP to the allosteric site of NAD-specific isocitrate dehydrogenase from bakers' yeast

The specificity of yeast NAD-specific isocitrate dehydrogenase for the structures of the allosteric effector 5'-AMP was examined with analogues modified in the purine ring, pentosyl group, and 5'-phosphate group. An unsubstituted 6-amino group was essential for activation as was the phosphoryl group at the 5'-position. Activity was retained when an oxygen function of the 5'-phosphoryl was replaced by sulfur (Murry & Atkinson, 1968) or by nitrogen (phosphoramidates). 2-NH2-AMP, 2-azido-AMP, and 8-NH2-AMP were active; 8-azido-AMP and 8-Br-AMP were inactive. The configuration or nature of substituents about carbons 2' and 3' of the pentosyl portion of AMP was not critical for allosteric activation since AMP analogues containing, e.g., 2',3'-dideoxyribose or the bulky 2',3'-O-(2,4,6-trinitrocyclo-hexadienylidene) substituent (TNP-AMP) were active. TNP-AMP was bound to the enzyme with fluorescence enhancement and had an S0.5 for activation similar to the S0.5 for AMP. Positive effector activity was decreased when the pentosyl moiety of 5'-AMP was replaced by the six-membered nitrogen-containing morpholine group, indicating that the pentosyl group may be critical as a spacer for the proper geometry of binding to enzyme at the 6-amino and 5'-phosphoryl groups of 5'-AMP. A comparison of molecular models of 5'-AMP with 8,5'-cycloAMP suggests that the species of 5'-AMP required for binding to the enzyme contains the purine and ribose moieties in an anti conformation and positioning of the 5'-phosphate trans with respect to carbon 4'.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies of ox-liver glutamate dehydrogenase oxidative deamination of two glutamate analogues, L-threo-gamma-methylglutamate and L-alpha-amino-gamma-nitraminobutyrate, in the presence of the allosteric effector ADP

Ox-liver glutamate dehydrogenase is known to utilise a wide range of amino acid substrates. Kinetic studies are presented here for L-threo-gamma-methylglutamate and L-alpha-amino-gamma-nitraminobutyrate in the presence of the allosteric effector ADP. The results presented are considered in the light of similar studies presented elsewhere in which the cofactor was systematically replaced by a variety of analogues. These amino acid analogues share the same pH optimum as glutamate, unlike the monocarboxylic amino acids including alanine and norvaline, and give linear double-reciprocal plots under the conditions used here. Studies with the alternative coenzymes have suggested an ordered addition of glutamate before coenzyme in the presence of ADP. The present results obtained under identical conditions support this conclusion.     

Inhibition of protohaem ferro-lyase by N-substituted porphyrins. Structural requirements for the inhibitory effect.

N-Methyl mesoporphyrin was a powerful inhibitor of protohaem ferro-lyase in vitro, whereas N-ethyl mesoporphyrin and N-methyl coproporphyrin were not and neither was the newly described green pigment produced by giving rats ethylene. This suggests that the size of the substituent at a pyrrole nitrogen and also the number of carboxylic acid side chains of the substituted porphyrin are important for the inhibitory effect. Evidence that N-methyl mesoporphyrin inhibited the enzyme, whereas the ethylene-derived pigment did not, was also obtained in vivo.     

Structural requirements for the inhibitory action of 12,13-epoxytrichothecenes on protein synthesis in eukaryotes

1. The inhibitory actions of ten trichothecene antibiotics were investigated, in reticulocyte cell-free systems synthesizing protein in vitro, by studying polyribosome profiles and kinetics of amino acid incorporation in the presence or absence of the drugs. 2. The modes of action observed were critically dependent on the drug concentrations used, but the antibiotics tested could be divided into four distinct groups, each exerting a characteristic inhibitory response. 3. The inhibitory action observed in every case was controlled by the chemical structure of the individual trichothecene and in particular was closely related to the nature of the substituent groups present on C-3, C-4, C-8 and C-15 of the molecule.     

Kinetic studies of glutamate dehydrogenase with glutamate and norvaline as substrates. Coenzyme activation and negative homotropic interactions in allosteric enzymes

1. Kinetic studies of glutamate dehydrogenase were made with wide concentration ranges of the coenzymes NAD(+) and NADP(+) and the substrates glutamate and norvaline. Initial-rate parameters were evaluated. 2. Deviations from Michaelis-Menten behaviour towards higher activity were observed with increasing concentrations of either coenzyme with glutamate as substrate, but not with norvaline as substrate. 3. In phosphate buffer, pH7.0, Lineweaver-Burk plots with either coenzyme as variable and a constant, large glutamate concentration showed three or four linear regions of different slope with relatively sharp discontinuities. Maximum rates obtained by extrapolation and Michaelis constants for the coenzymes increased in steps with increase of coenzyme concentration. 4. In the absence of evidence of heterogeneity of the enzyme and coenzyme preparations, the results are interpreted in terms of negative homotropic interactions between the enzyme subunits. It is suggested that sharp discontinuities in Lineweaver-Burk plots or reciprocal binding plots may be characteristic of this new type of interaction, which can be explained in terms of an Adair-Koshland model, but not by the model of Monod, Wyman & Changeux.     

Structural requirements of para-alkylphenols to bind to estrogen receptor.

Octyl- and nonylphenols in the environment have been proposed to function as estrogens. To gain insight into their structural essentials in binding to the estrogen receptor, a series of phenols with saturated alkyl groups at the para position, HO-C6H4-CnH2n+1 (n = 0-12), were examined for their ability to displace [3H]17beta-estradiol in the recombinant human estrogen receptor, which was expressed in Sf9 cells using the vaculovirus expression system. All tested para-alkylphenols were found to bind fully to the estrogen receptors in a dose-dependent manner. The interaction of alkylphenols with the receptor became stronger with increase in the number of the alkyl carbons and the activity was maximized with n = 9 of nonylphenol. Phenol (n = 0) exhibited weak but full binding to the receptor, whereas anisole with a protected phenolic hydroxyl group was completely inactive. Also, alkanes such as n-octane, 2,2, 4-trimethylpentane corresponding to tert-octane, and n-nonane exhibited no binding. The results indicate that the binding of para-alkylphenols to the estrogen receptor is due to the effect of covalent bonding of two constituents of the phenol and alkyl groups, which correspond to the A-ring and hydrophobic moiety of the steroid structure, respectively. When alkylphenols were examined for their receptor binding conformation by 1H-NMR measurements and ab initio molecular orbital calculations, it was suggested that nonbranched alkyl groups are in an extended conformation, while branched alkyl groups are in a folded conformation. These results suggest that branched and nonbranched alkyl moieties of alkylphenols interact differently with the lipophilic ligand binding cavity of the estrogen receptor when compared to the binding of 17beta-estradiol.     

Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action

To clarify the structure-activity relationships of flavonoids for nitric oxide (NO) production inhibitory activity, we examined the inhibitory effects of 73 flavonoids on NO production in lipopolysaccharide-activated mouse peritoneal macrophages. Among those flavonoids, apigenin (IC(50)=7.7 microM), diosmetin (8.9 microM), and tetra-O-methylluteolin (2.4 microM), and hexa-O-methylmyricetin (7.4 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the activities of flavones were stronger than those of corresponding flavonols; (2) the glycoside moiety reduced the activity; (3) the activities of flavones were stronger than those of corresponding flavanones; (4) the flavones and flavonols having the 4'-hydroxyl group showed stronger activities than those lacking the hydroxyl group at the B ring and having the 3',4'-dihydroxyl group; (5) the flavonols having the 3',4'-dihydroxyl group (catechol type) showed stronger activities than those having the 3',4',5'-trihydroxyl group (pyrogallol type); (6) the 5-hydroxyl group tended to enhance the activity; (7) methylation of the 3-, 5-, or 4'-hydroxyl group enhanced the activity; (8) the activities of isoflavones were weaker than those of corresponding flavones; (9) methylation of the 3-hydroxyl group reduced the cytotoxicity. In addition, potent NO production inhibitors were found to inhibit induction of inducible nitric oxide synthase (iNOS) without iNOS enzymatic inhibitory activity.