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PubMed1980

Glutamate dehydrogenase deficiency in three patients with spinocerebellar syndrome.

Four nicotinamide-adenine dinucleotide phosphate-requiring enzymes were measured in disrupted cultured skin fibroblasts from a 19-year-old patient with juvenile onset of a spinocerebellar and extrapyramidal syndrome. There was marked reduction in the activity of glutamate dehydrogenase (GDH) (22% of mean control activity); GDH activity was also decreased in homogenates of leukocytes from this patient (38% of mean control activity). GDH activity was measured in the leukocytes of two siblings afflicted with adult-onset spinocerebellar syndrome and found to be decreased in both (29% and 31% of mean control activity); an unaffected sibling had normal GDH activity. Mixing experiments with control fibroblast and leukocyte homogenates did not show the presence of a GDH inhibitor in cells from these patients. This allosterically regulated enzyme was stimulated by adenosine 5'-diphosphate (10(-3) M) and inhibited by guanosine 5'-triphosphate (10(-3) M) in both fibroblast and leukocyte homogenates; these changes occurred in equal proportions in the patients and controls. The decreased fibroblast and leukocyte GDH activity persisted at different concentrations of the enzyme's substrates and with successive passages of cultured fibroblasts. GDH may have an important role in the metabolism of glutamate, a putative neurotransmitter in cerebellum, brainstem, and spinal cord. A genetic deficiency of GDH may underlie some forms of spinocerebellar ataxias.

Adult↗
PubMed2003

Phosphoribosylpyrophosphate synthetase overactivity as a cause of uric acid overproduction in a young woman.

Overactivity of phosphoribosylpyrophosphate synthetase (PRS) is an X chromosome-linked disorder of purine metabolism that is characterized by gout with uric acid overproduction and, in some families, neurodevelopmental impairment. We present the case of a 24-year-old Spanish woman with renal colic and hyperuricemia, which first manifested at age 11 years. Results of enzymatic and genetic studies supported the view that accelerated purine nucleotide and uric acid production in this woman resulted from defective allosteric regulation of PRS activity, which is, in turn, a consequence of a mutation in one of the patient's PRPS1 genes: an A-to-T substitution at nucleotide 578, encoding leucine for histidine at amino acid residue 192 of the mature PRS1 isoform. A previous example of disordered regulation of PRS1 activity in a family with a different substitution at the same amino acid residue strengthens this proposed mechanism. This is the first reported instance of PRS overactivity in which the propositus and sole affected family member is a woman.

Adult↗
PubMed1989

How does a key fit a flexible lock? Structure and dynamics in receptor function.

The preceding five years have brought remarkable advances in our understanding of the primary structure of drug receptors. The roles of certain amino acid residues in binding drugs and effecting receptor function have been proposed. As even more detailed structures become available, the goal of rational design of drug molecules based on predicted fits between the drug and its receptor will be near at hand. Although none of the classical receptors has yet yielded to X-ray crystallographic analysis, the methods of molecular biology facilitate the production of the large amounts of these rare proteins necessary for crystallization. Receptor proteins share one fundamental characteristic with allosterically regulated enzymes. Both have the structural flexibility that allows information to be transmitted to distant parts of the molecule. We will discuss recent observations about receptor structure and the dynamic nature of drug receptors, and pose questions about the significance of receptor dynamics for drug design.

Amino Acid Sequence↗
PubMed1998

Ribonucleotide reductase in Bacillus subtilis--evidence for a Mn-dependent enzyme.

The reduction of 2'-ribonucleotides to 2'-deoxyribonucleotides, a unique step in DNA formation, is catalyzed by ribonucleotide reductase (RRase), an allosterically regulated, cell cycle-dependent enzyme. This work reports a reversible impairment of DNA formation and ribonucleotide reduction upon manganese depletion in Bacillus subtilis demonstrated through in vivo labeling with necleic acid precursors and enzyme assays with ether-permeabilized cells. No deoxyadenosylcobalamin-dependent reduction of ribonucleotides was detected in the cytosol, and the properties of a partially purified enzyme fraction, i.e., sensitivity towards EDTA and hydroxyurea (HU), indicated a metal-dependent type of RRase. The enzyme was enriched by gel filtration on Superose 12 from glycerol- or fumarate-grown cells and submitted to Q-band electron paramagnetic resonance (EPR) spectroscopy for further characterization of the metal center. A distinct Mn(II) signal was obtained in both preparations characteristic of a protein-bound mangaenese in a mononuclear metal center with axial symmetry. The intensity of this Mn signal was not affected by addition of the radical scavenger HU (10 mM) but reduced in the presence of 2.5 mM EDTA. On the basis of these results, we suggest that Bacillus subtilis has a Mn-dependent ribonucleotide reductase.

Bacillus subtilis↗
PubMed2003

Structural alterations of enkephalins in the presence of GM1 ganglioside micelles.

The enkephalins are endogenous neurotransmitters and bind with high affinity at the delta-receptor. Gangliosides, the major glycans of nerve cells, known to interact both with receptors and ligands on the cell surface, have been implicated to modulate the actions of opioid receptors by allosteric regulation (Wu, G.; Lu, Z. H.; Wei, T. J.; Howells, R. D.; Christoffers, K.; Leeden R. W. Ann NY Acad Sci 1998, 845, 126-138). We have studied the interactions between enkephalins and monosialylated ganglioside GM1 using NMR spectroscopy and fluorescence. The structural models of enkephalins in the presence of GM1 micelles were generated using two-dimensional (1)H-ROESY experiments along with restrained molecular dynamics simulations. We report a conformational alteration of enkephalins in the presence of GM1 micelles.

Animals↗
PubMed1989

Human atrial natriuretic peptide receptor defines a new paradigm for second messenger signal transduction.

We isolated cDNAs encoding a 115 kd human atrial natriuretic peptide (alpha ANP) receptor (ANP-A receptor) that possesses guanylate cyclase activity, by low-stringency hybridization with sea urchin Arbacia punctulata membrane guanylate cyclase probes. The human ANP-A receptor has a 32 residue signal sequence followed by a 441 residue extracellular domain homologous to the 60 kd ANP-C receptor. A 21 residue transmembrane domain precedes a 568 residue cytoplasmic domain with homology to the protein kinase family and to a subunit of the soluble guanylate cyclase. COS-7 cells transfected with an ANP-A receptor expression vector displayed specific [125I]alpha ANP binding, and exhibited alpha ANP stimulated cGMP production. These data demonstrate a new paradigm of cellular signal transduction where extracellular ligand binding allosterically regulates cyclic nucleotide second-messenger production by a receptor cytoplasmic catalytic domain.

Amino Acid Sequence↗
PubMed1992

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.

Allosteric Regulation↗
PubMedDate not supplied

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant.

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR. The oxidation of [1,2,3-13C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3-13C]propionate or [2-13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.

Allosteric Regulation↗
PubMedDate not supplied

Kinetic analysis and modelling of the allosteric behaviour of liver and muscle glycogen phosphorylases.

Allosteric enzymes have very complex kinetic behaviours which are primarily interpreted through simplified models. To describe the functional properties of liver and muscle glycogen phosphorylase isozymes we have developed an experimental strategy based on the measurements of initial reaction rates in the presence of different concentrations of the effectors glucose-1-phosphate and methyl-xanthines. Using the extensive structural information available for the two glycogen phosphorylase conformers T (inactive) and R (active) with different ligands, we have applied the Monod-Wyman-Changeux model and analysed the results in the context of the exclusive binding of the inhibitors to the T state, meanwhile the substrate glucose-1-phosphate binds to both, the R and T states. The kinetic analysis shows a good agreement between our model and the results obtained from the glycogen phosphorylases and inhibitors included in this study, which demonstrates the validity of the approach described here.

Allosteric Regulation↗
PubMed1976

Lipid-protein interactions: enhancement of enzyme activity of L-glutamic acid dehydrogenase by nonionic detergents.

Five nonionic detergents enhanced the activity of L-glutamic acid dehydrogenase [L-glutamate:nicotinamide adenine dinucleotide phosphate oxidoreductase (deaminating) (EC 1.4.1.3)]. These detergents activated the enzyme toward alpha-ketoglutaric acid reduction, causing a decrease in the sensitivity of the enzyme to allosteric regulation by guanosine 5-triphosphate. There was also a diminution of the enhancing effect of the modifier adenosine 5-diphosphate on the enzyme's L-glutamic acid dehydrogenase activity. These detergents may cause a conformational change in the enzyme, and this change could lead to an increase in the binding of the substrates for the alpha-ketoglutaric acid reduction. Accompanied with this conformational change would be a decrease in the binding of the modifier guanosine 5'-triphosphate, with no concomitant change in the binding of the adenosine 5'-diphosphate modifier.

Adenosine Diphosphate↗
PubMed1991

Use of a human serum albumin-based high-performance liquid chromatography chiral stationary phase for the investigation of protein binding: detection of the allosteric interaction between warfarin and benzodiazepine binding sites.

The interaction between the benzodiazepine and the warfarin binding sites in human serum albumin (HSA) has been investigated using an HSA-based HPLC chiral stationary phase (HSA-CSP). (R)-Warfarin and (S)-warfarin were added to the mobile phase and racemic mixtures of oxazepam, lorazepam, and their hemisuccinic derivatives were injected onto the HSA-CSP. The presence of (R)-warfarin in the mobile phase did not significantly affect the chromatographic retention (expressed as capacity factor, k') of the investigated benzodiazepine hemisuccinate derivatives. The presence of (S)-warfarin did not significantly affect the k' of oxazepam and oxazepam hemisuccinate, but resulted in a dramatic increase in the k' of (S)-lorazepam hemisuccinate and also improved the enantiomeric resolution of lorazepam. These results confirm the existence of an allosteric interaction between the benzodiazepine binding site and the warfarin binding site. Furthermore, the study indicates that chromatography on the silica-immobilized HSA can detect interactions between binding sites on the protein. This can be of great importance in the determination of drug-drug interactions.

Allosteric Regulation↗
PubMed2003

Cholinergic nicotinic receptors: competitive ligands, allosteric modulators, and their potential applications.

Discovery of the important role played by nicotinic acetylcholine receptors (nAChRs) in several CNS disorders has called attention to these membrane proteins and to ligands able to modulate their functions. The existence of different subtypes at multiple levels has complicated the understanding of this receptor's physiological role, but at the same time has increased the efforts to discover selective compounds in order to improve the pharmacological characterization of this kind of receptor and to make the possible therapeutical use of its modulators safer. This review focuses on the structure of new ligands for nAChRs, agonists, antagonists and allosteric modulators, and on their possible applications.

Allosteric Regulation↗
PubMed1993

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.

Allosteric Regulation↗
PubMed1994

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.

Amino Acid Sequence↗
PubMed1994

Glu-50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure.

Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity high-affinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu-50 (Glu-50-->Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme. In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme. However, the Glu-50-->Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steady-state kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50-->Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.

Alanine↗
PubMed2004

Is allostery an intrinsic property of all dynamic proteins?

Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.

Allosteric Regulation↗
PubMed1996

Differential effects of bovine PA28 on six peptidase activities of the lobster muscle proteasome (multicatalytic proteinase).

PA28, an endogenous activator of the bovine proteasome, stimulated the branched-chain amino acid-preferring (BrAAP; 99-fold activation), small-neutral amino acid-preferring (11-fold), acidic chymotrypsin-like (26-fold), and peptidylglutamyl peptide hydrolase (14-fold) activities of the lobster muscle proteasome, while having little or no effect on the trypsin-like, neutral chymotrypsin-like, and caseinolytic activities. These results show that the BrAAP activity, which has been linked to the degradation of myofibrillar proteins by the heat-activated proteasome, is allosterically regulated. However, the activation by PA28 differs from that induced by heat treatment, since heat activation stimulated both the BrAAP and the proteolytic activities but not the other peptidase activities. PA28 shifted the pH optimum of the acidic chymotrypsin-like activity from pH 6-6.5 to pH 7-7.5, while stimulating the activity about 10-fold. These results suggest that PA28 is involved in the activation of the acidic chymotrypsin-like component at physiological pH.

Amino Acids↗