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PubMed2000

Regulation of human D(1), d(2(long)), d(2(short)), D(3) and D(4) dopamine receptors by amiloride and amiloride analogues.

1. The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D(1), D(2), D(3) and D(4) dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D(3) subtype for acceleration of radioligand dissociation. 3. In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D(1), D(2(short)), D(2(long)) and D(3) receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4. At the D(4) subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D(4) subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5. MIA reduced the potency with which dopamine stimulated [(35)S]-GTPgammaS binding at the D(2) receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

Allosteric Regulation↗
PubMed2002

Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway.

The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.

Allosteric Regulation↗
PubMed1997

Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity.

Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-expressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both N- and C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNOS in endothelial cells, therefore, may involve not only positive allosteric regulation by Ca2+/CaM, but also negative allosteric regulation by caveolin-1.

Amino Acid Sequence↗
PubMed2002

Cell cycle-dependent regulation of pyrimidine biosynthesis.

De novo pyrimidine biosynthesis is activated in proliferating cells in response to an increased demand for nucleotides needed for DNA synthesis. The pyrimidine biosynthetic pathway in baby hamster kidney cells, synchronized by serum deprivation, was found to be up-regulated 1.9-fold during S phase and subsequently down-regulated as the cells progressed through the cycle. The nucleotide pools were depleted by serum starvation and were not replenished during the first round of cell division, suggesting that the rate of utilization of the newly synthesized nucleotides closely matched their rate of formation. The activation and subsequent down-regulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian pyrimidine biosynthesis. As the culture approached S-phase there was an increased sensitivity to the allosteric activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP inhibition, changes that were reversed when cells emerged from S phase. The allosteric regulation of CAD is known to be modulated by MAP kinase (MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as by autophosphorylation. CAD was found to be fully autophosphorylated in the synchronized cells, but the level remained invariant throughout the cycle. Although the MAPK activity increased early in G(1), the phosphorylation of the CAD MAPK site was delayed until just before the onset of S phase, probably due to antagonistic phosphorylation by PKA that persisted until late G(1). Once activated, pyrimidine biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase. Thus, the cell cycle-dependent regulation of pyrimidine biosynthesis results from the sequential phosphorylation and dephosphorylation of CAD under the control of two important signaling cascades.

Animals↗
PubMed2004

Evolution of glutamate dehydrogenase regulation of insulin homeostasis is an example of molecular exaptation.

Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the oxidative deamination of glutamate to 2-oxoglutarate. While this enzyme does not exhibit allosteric regulation in plants, bacteria, or fungi, its activity is tightly controlled by a number of compounds in mammals. We have previously shown that this regulation plays an important role in insulin homeostasis in humans and evolved concomitantly with a 48-residue "antenna" structure. As shown here, the antenna and some of the allosteric regulation first appears in the Ciliates. This primitive regulation is mediated by fatty acids and likely reflects the gradual movement of fatty acid oxidation from the peroxisomes to the mitochondria as the Ciliates evolved away from plants, fungi, and other protists. Mutagenesis studies where the antenna is deleted support this contention by demonstrating that the antenna is essential for fatty acid regulation. When the antenna from the Ciliates is spliced onto human GDH, it was found to fully communicate all aspects of mammalian regulation. Therefore, we propose that glutamate dehydrogenase regulation of insulin secretion is a example of exaptation at the molecular level where the antenna and associated fatty acid regulation was created to accommodate the changes in organelle function in the Ciliates and then later used to link amino acid catabolism and/or regulation of intracellular glutamate/glutamine levels in the pancreatic beta cells with insulin homeostasis in mammals.

Adenosine Diphosphate↗
PubMedDate not supplied

[Comparative theoretic analysis of an open reaction S1 goes to and comes from S2 E(R,T) in which the oligomeric enzyme E(R,T) is isosterically or allosterically activated by the product S2].

In connection with evolutionary aspects of the mechanisms of allosteric regulation of cell metabolism, a mathematical model of an open reaction leads to S1 E(R,T) in equilibrium S2 leads to involving product activation of the olygomeric enzyme E(R,T) whose protomers undergo the concerted conformational transitions R in equilibrium T, has been analysed. Two activation mechanisms, isosteric and allosteric, were considered. Both mechanisms produce qualitatively the same effects: the input characteristic of the reaction possesses hysteresis, which causes multiple steady states and self-oscillations. In the case of isosteric activation, only a small fraction of the maximum enzyme activity is utilized because of the strong competition between S1 and S2 for the active sites. In the case of allosteric regulation, this competition may almost completely be eliminated and the enzyme activity may be utilized with high efficiency, provided the affinity of S2 for the allosteric sites is at least an order of magnitude higher than for the active sites. Qualitative similarity between the two cases of product activation, in spite of their great quantitative discrepancy, favours the hypothesis that the mechanisms of isosteric regulation were the direct precursors of the homological allosteric regulatory mechanisms.

Allosteric Regulation↗
PubMed2003

Molecular design and functional organization of the RecA protein.

The bacterial RecA protein participates in a remarkably diverse set of functions, all of which are involved in the maintenance of genomic integrity. RecA is a central component in both the catalysis of recombinational DNA repair and the regulation of the cellular SOS response. Despite the mechanistic differences of its functions, all require formation of an active RecA/ATP/DNA complex. RecA is a classic allosterically regulated enzyme, and ATP binding results in a dramatic increase in DNA binding affinity and a cooperative assembly of RecA subunits to form an ordered, helical nucleoprotein filament. The molecular events that underlie this ATP-induced structural transition are becoming increasingly clear. This review focuses on descriptions of our current understanding of the molecular design and allosteric regulation of RecA. We present a comprehensive list of all published recA mutants and use the results of various genetic and biochemical studies, together with available structural information, to develop ideas regarding the design of RecA functional domains and their catalytic organization.

Allosteric Regulation↗
PubMed1995

Pleiotropic regulation of central carbohydrate metabolism in Escherichia coli via the gene csrA.

The carbon storage regulator gene csrA has been shown previously to dramatically affect the biosynthesis of intracellular glycogen in Escherichia coli through its negative control of the expression of two glycogen biosynthetic operons and the gluconeogenic gene pckA (Romeo, T., Gong, M., Liu, M. Y., and Brun-Zinkernagel, A. M. (1993) J. Bacteriol. 175, 4744-4755). Examination of the effects of csrA on several enzymes, genes, and metabolites of central carbohydrate metabolism now establishes a more extensive role for csrA in directing intracellular carbon flux. Phosphoglucomutase and the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate synthetase were found to be under the negative control of csrA, and these enzyme activities were maximal during the early stationary phase of growth. The enzymes glucose-6-phosphate isomerase, triose-phosphate isomerase, and enolase were positively regulated by csrA. Thus, csrA exerts reciprocal effects on glycolysis versus gluconeogenesis and glycogen biosynthesis. The glycolytic isozymes pyruvate kinase F and A (encoded by pykF and pykA, respectively) and phosphofructokinase I and II (pfkA and pfkB, respectively) exhibited differential regulation via csrA. Since the individual members of these isozyme pairs are allosterically regulated by different cellular metabolites, csrA is also capable of fine-tuning the allosteric regulation of glycolysis. In contrast, the expression of genes of the pentose phosphate pathway was weakly or negligibly affected by csrA.

Bacterial Proteins↗
PubMed1998

N- and C-terminal peptide sequences are essential for enzyme assembly, allosteric, and/or catalytic properties of ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase is a key regulatory enzyme in starch synthesis in most plant tissues. Unlike the allosteric regulatory dependent properties of the leaf enzyme, the enzymes from non-photosynthetic tissues exhibit varying levels of sensitivity to allosteric regulation, a behavior which may be an inherent property of the enzyme or a product of post-translational modification. As partial proteolysis of the holoenzyme may account for the wide variation of allosteric regulatory behavior exhibited by enzymes from non-photosynthetic tissues, small N- and C-terminal peptide deletions were made on either the potato large and small subunit and co-expressed with the counterpart wild-type subunit in Escherichia coli. Removal of the putative carboxy-terminal allosteric binding region from either subunit type results in an abolishment of enzyme formation indicating that the carboxy terminus of each subunit type is essential for proper subunit folding and/or enzyme assembly as well as its suggested role in allosteric regulation. Removal of a small 10 amino acid peptide from the N-terminus of the small subunit increased its resistance to the allosteric inhibitor Pi as well as its sensitivity to heat treatment. Likewise, removal of the corresponding peptide (17 residues) at the N-terminus of the large subunit also increased its resistance towards Pi inhibition but, in addition, increased its sensitivity to 3-PGA activation. Deletion of an additional 11 residues reversed these changes in allosteric properties but at the expense of a reduced catalytic turnover rate. Combined, these results indicate that the N- and C-terminal regions are essential for the proper catalytic and allosteric regulatory properties of the potato ADP-glucose pyrophosphorylase. The possible significance of these results on the observed insensitivity to effector molecules by ADP-glucose pyrophosphorylases from other non-photosynthetic tissues is discussed.

Allosteric Regulation↗
PubMed2002

Transplanting allosteric control of enzyme activity by protein-protein interactions: coupling a regulatory site to the conserved catalytic core.

Glycerol kinase from Escherichia coli, but not Haemophilus influenzae, is inhibited allosterically by phosphotransferase system protein IIA(Glc). The primary structures of these related kinases contain 501 amino acids, differing at 117. IIA(Glc) inhibition is transplanted from E. coli glycerol kinase into H. influenzae glycerol kinase by interconverting only 11 of the differences: 8 residues that interact with IIA(Glc) at the allosteric binding site and 3 residues in the conserved ATPase catalytic core that do not interact with IIA(Glc) but the solvent accessible surface of which decreases when it binds. The three core residues are crucial for coupling the allosteric site to the conserved catalytic core of the enzyme. The site of the coupling residues identifies a regulatory locus in the sugar kinase/heat shock protein 70/actin superfamily and suggests relations between allosteric regulation and the active site closure that characterizes the family. The location of the coupling residues provides empirical validation of a computational model that predicts a coupling pathway between the IIA(Glc)-binding site and the active site [Luque, I. & Freire, E. (2000) Proteins Struct. Funct. Genet. Suppl. 4, 63-71]. The requirement for changes in core residues to couple the allosteric and active sites and switching from inhibition to activation by a single amino acid change are consistent with a postulated mechanism for molecular evolution of allosteric regulation.

Allosteric Regulation↗
PubMed2001

Modelling of promiscuous receptor-Gi/Gs-protein coupling and effector response.

A single G-protein-coupled receptor might activate multiple G-protein species. This multiplex coupling ability can be used by tissues to regulate signalling; for the pharmacologist, such multiplex coupling might cause difficulties in the interpretation of experimental data. In this article, we present mathematical models for the activation of two separate G-protein species by a single receptor. Issues addressed concern mutual antagonism between the G proteins and the availability of an already activated receptor for interaction with a new G protein (receptor-G-protein-effector complexing versus free diffusion of G proteins) in addition to receptor-G-protein precoupling at different G-protein and receptor expression levels. The output from the receptor models uses, as readout, a new model for adenylyl cyclase regulation by two allosteric regulators (i.e. G(s) and G(i)).

Animals↗
PubMed2000

Regulation of glycogen synthase. Identification of residues involved in regulation by the allosteric ligand glucose-6-P and by phosphorylation.

The major yeast glycogen synthase, Gsy2p, is inactivated by phosphorylation and activated by the allosteric ligand glucose-6-P. From studies of recombinant proteins, the control can be accommodated by a three-state model, in which unphosphorylated enzyme has intermediate activity (state II). Glucose-6-P increased V(max)/K(m) by about 2-fold (state III), whereas phosphorylation by the cyclin-dependent protein kinase Pcl10p/Pho85p decreased V(max)/K(m) by approximately 30-fold (state I). In the presence of glucose-6-P, state III is achieved regardless of phosphorylation state. The enzyme forms complexes in solution with the yeast glycogenin Glg2p, but this interaction appears not to affect control either by glucose-6-P binding or by phosphorylation. Scanning mutagenesis was applied to identify residues potentially involved in ligand binding. Of 22 mutant enzymes analyzed, seven were essentially inactive. Five mutant proteins were altered in their activation by glucose-6-P, and two were completely unaffected by the hexose phosphate. One of these, R586A/R588A/R591A (all three of the indicated Arg residues mutated to Ala), had wild-type activity and was normally inactivated by phosphorylation. A second mutant, R579A/R580A/R582A, had somewhat reduced V(max), but its activity was not greatly reduced by phosphorylation. The Arg residues in these two mutants are restricted to a highly conserved, 13-residue segment of Gsy2p that we propose to be important for glucose-6-P binding and/or the ability of the enzyme to undergo transitions between activity states.

Allosteric Regulation↗
PubMed2001

Regulation of E. coli glycogen phosphorylase activity by HPr.

Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases.

Amino Acid Sequence↗
PubMed2002

Multiple conformational changes in enzyme catalysis.

Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.

Allosteric Regulation↗
PubMed1998

Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa.

The catalytic activity of the trypsin-like serine protease coagulation factor VIIa is allosterically regulated. In this work, we employed monoclonal antibodies as probes to analyze conformational changes in the VII protease domain that are induced by zymogen activation, cofactor tissue factor (TF) binding, and active site occupancy. The epitopes of three monoclonal antibodies were mapped using a panel of 57 individual alanine replacement mutants in the protease domain. Two of the antibodies had typical "hot spot" epitopes in a basic cluster above the active site cleft and antibody binding to these epitopes was not affected by zymogen activation, TF binding, or active site occupancy. In contrast, the binding kinetics of VII/VIIa to a monoclonal antibody that mapped to an extended epitope overlapping with the macromolecular substrate exosite was affected by each of the conformational transitions of the VIIa protease domain. The changes in antibody affinity are consistent with a transition from zymogen VII to the TF.VIIa complex, with free enzyme VIIa as an intermediate that retains some zymogen-like features responsible for its low catalytic activity. In contrast, active site occupancy resulted in effects that were qualitatively different from the effects of zymogen activation on the antibody epitope. This provides novel insight into the conformational interdependence between the active site, the region for macromolecular substrate recognition, and the cofactor binding exosite of this allosterically regulated serine protease.

Allosteric Regulation↗
PubMed2001

Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions.

Yeast phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1) are allosterically regulated enzymes that catalyse essentially irreversible reactions in glycolysis. Both the synthesis and activity of these enzymes are tightly regulated. To separate experimentally the control of Pf1k and Pyk1 synthesis from their allosteric regulation, a congenic set of PFK1, PFK2 and PYK1 mutants was constructed in which these wild-type coding regions were driven by alternative promoters. Mutants carrying PGK1 promoter fusions displayed normal rates of growth, glucose consumption and ethanol production, indicating that the relatively tight regulation of Pyk1 and Pf1k synthesis is not essential for glycolytic control under fermentative growth conditions. Mutants carrying fusions to an enhancer-less version of the PGK1 promoter (PGK1(Delta767)) expressed Pyk1 and Pf1k at about 2.5-fold lower levels than normal. Physiological and metabolic analysis of the PFK1 PFK2 double mutant indicated that decreased Pf1k had no significant effect on growth, apparently due to compensatory increases in its positive effector, fructose 2,6-bisphosphate. In contrast, growth rate and glycolytic flux were reduced in the PGK1(Delta767)-PYK1 mutant, which had decreased Pyk1 levels. Unexpectedly, the reduced Pyk1 levels caused the flow of carbon to the TCA cycle to increase, even under fermentative growth conditions. Therefore, Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast.

Fermentation↗