Allosterically

Department of Chemistry ‘IFM’, University of Torino, Italy; 2Department of Biology, University ‘Roma Tre’, Rome, Italy; 3Department of Structural and Functional Biology, University of Insubria, Italy Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem –human serum albumin (ferric HSA–haem) and of ferrous nitrosylated haem –human serum albumin (ferrous HSA –haem-NO) is reported.

7p system191 01-06-2013 46 3   Download

Nitric oxide (NO), the principal endogenous ligand for soluble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO. Previous studies demonstrated that 2-substituted adenine nucleotides, including 2-methylthioATP (2MeSATP) and 2-chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane-bound receptor for the Escherichia coli heatstable enterotoxin in the intestine. The present study examined the effects of 2-substituted adenine nucleotides on crude and purified sGC. ...

8p research12 01-06-2013 36 4   Download

Fructose 2,6-bisphosphate (Fru-2,6-P2 ) plays an important role in the regulation of major carbohydrate fluxes as both allosteric activator and inhibitor of target enzymes. To examine the role of Fru-2,6-P2in the regulation of hepatic carbohydrate metabolism in vivo,Fru-2,6-P2 levels were elevated in ADM mice with adenovirus-mediated overex-pression of a double mutant bifunctional enzyme, 6-phos-phofructo-2-kinase/fructose-2,6-bisphosphatase (n¼6), in comparison to normal control mice (control,n¼6). ...

9p research12 29-04-2013 50 5   Download

CTP synthase catalyzes the reaction glutamine + UTP +ATPfiglutamate + CTP + ADP + Pi.The rate of the reaction is greatly enhanced by the allosteric activator GTP.We have studied the glutaminase half-reactionofCTP synthase from Lactococcus lactisand its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia colienzyme, GTP activation of the L. lactis enzyme did not result in similarkcat values for the glutami-nase activity and glutamine hydrolysis coupled to CTP synthesis. ...

8p research12 29-04-2013 44 2   Download

Escherichia coli 3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in serine biosynthesis, and is allosterically inhibitedby serine. Structural studies revealeda homotetramer in which the quaternary arrangement of subunits formed an elongated ellipsoid. Each subunit consisted of three domains: nucleotide, substrate and regu-latory. InPGDH, extensive interactions are formedbetween nucleotide binding domains.

9p research12 23-04-2013 36 2   Download

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes inEscheri-chia coli. As binding of cAMP leads to undefined conform-ational changes in CRP, we performed a steady-state and time-resolved fluorescence study to showhow the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single trypto-phan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached toCys178.

11p tumor12 20-04-2013 31 3   Download

Multiple signalling pathways are involved in themechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphory-lase (CP-91149) that causes dephosphorylation of phos-phorylasea, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phos-phorylaseaas alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes.

9p fptmusic 16-04-2013 41 3   Download

Cytidine 5¢-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia orL-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed muta-genesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coliCTP synthase thatwere highly susceptible to proteolysis. ...

12p fptmusic 16-04-2013 39 4   Download

The effect of CoA on the characteristic light decay of the firefly luciferase catalysed bioluminescence reaction was studied. At least part of the light decay is due to the luciferase catalysed formation of dehydroluciferyl-adenylate (L-AMP), a by-product that results from oxidation of luciferyl-adenylate (LH2-AMP), and is a powerful inhibitor of the bioluminescence reaction (IC50¼6nm).

11p fptmusic 12-04-2013 40 4   Download

Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myri-state binding to the N and B conformational states of Mn(III)heme–HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal....

12p fptmusic 12-04-2013 45 4   Download

This work proposes a model of the metabolic branch-point between the methionine and threonine biosynthesis path-ways inArabidopsis thalianawhich involves kinetic compe-tition for phosphohomoserine between the allosteric enzyme threonine synthase and the two-substrate enzyme cysta-thionine c-synthase. Threonine synthase is activated by S-adenosylmethionine and inhibited by AMP. Cystathio-ninec-synthase condenses phosphohomoserine to cysteine via a ping-pong mechanism.

13p fptmusic 12-04-2013 39 3   Download

Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy meta-bolism in these protozoan parasites. A 6-phosphofructo-2-kinase, respon-sible for the synthesis of this regulator, was highly purified from the bloodstream form ofTrypanosoma bruceiand kinetically characterized. By searching trypanosomatid genome databases, four genes encoding proteins homologous to the mammalian bifunctional enzyme 6-phosphofructo-2-kinase⁄fructose-2,6-bisphosphatase (PFK-2⁄FBPase-2) were found for both T. ...

19p fptmusic 11-04-2013 32 3   Download

Whereas most membrane receptors are oligomeric entities, G-protein-coupled receptors have long been thought to function as monomers. Within the last 15 years, accumulating data have indicated that G-protein-coupled receptors can form dimers or even higher ordered oligomers, but the gen-eral functional significance of this phenomena is not yet clear.

9p fptmusic 11-04-2013 37 3   Download

Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi Insights into cooperative and allosteric mechanisms Sigrid Van Boxstael1, Dominique Maes2,3 and Raymond Cunin1 1 Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, Belgium 2 Ultrastructuur, Vrije Universiteit Brussel, Belgium 3 Vlaams Interuniversitair Instituut voor Biotechnologie, Belgium Keywords allostery; aspartate transcarbamylase; cooperativity; inhibition by analogues; Pyrococcus abyssi Correspondence R.

14p awards 06-04-2013 42 3   Download

Theuppgene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed inEscherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60
C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP.

14p awards 06-04-2013 24 5   Download

Here we examine the enantioselectivity of the allosteric and substrate bind-ing sites of murine ribonucleotide reductase (mRR). l-ADP binds to the active site andl-ATP binds to both the s- and a-allosteric sites of mR1 with affinities that are only three- to 10-fold weaker than the values for the corresponding d-enantiomers. These results demonstrate the potential of l-nucleotides for interacting with and modulating the activity of mRR, a cancer chemotherapeutic and antiviral target.

0p awards 06-04-2013 38 2   Download

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes inEscherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-ter-minal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein.

14p awards 05-04-2013 42 4   Download

GTP is an allosteric activator of CTP synthase and acts to increase thekcat for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase.

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Temperature effects on the kinetic properties of phosphofructokinase (PFK) purified from skeletal muscle of the golden-mantled ground squirrel, Spermophilus lateralis, were examined at 37
C and 5
C, values character-istic of body temperatures in euthermia vs. hibernation. The enzyme showed reduced sensitivity to all activators at 5
C, theKa values for AMP, ADP, NH4 + and F2,6P2 were 3–11-fold higher at 5
C than at 37
C. Inhibition by citrate was not affected whereas phosphoenolpyruvate, ATP and urea became more potent inhibitors at low temperature. ...

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This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfishHoplosternum littorale, a species that displays facultative accessorial air oxygen-ation. We demonstrate that water potential affects the oxy-gen affinity ofH. littoraleHbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize thehigh-affinitystateof theHb.

0p awards 05-04-2013 41 3   Download