168 results on '"Paul, V."'
Search Results
2. Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen
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Gabriel Birrane, Francisco Javier Cañada, Helena Coelho, Paul V. Murphy, J.G. Luz, Adele Gabba, Francisco Corzana, Ana Diniz, Agnieszka Bogucka, Filipa Marcelo, Gabrielle’s Angel Foundation for Cancer Research, Science Foundation Ireland, Irish Research Council, Fundação para a Ciência e a Tecnologia (Portugal), Agencia Estatal de Investigación (España), Instituto de Salud Carlos III, Gabba, Adele, Bogucka, Agnieszka, Luz, John G., Diniz, Ana, Coelho, Helena, Corzana, Francisco, Cañada, F. Javier, Marcelo, Filipa, Murphy, Paul V., Birrane, Gabriel, Gabba, Adele [0000-0001-8240-6482], Bogucka, Agnieszka [0000-0001-5317-4344], Luz, John G. [0000-0001-7651-2094], Diniz, Ana [0000-0003-1698-4668], Coelho, Helena [0000-0002-4630-4259], Corzana, Francisco [0000-0001-5597-8127], Cañada, F. Javier [0000-0003-4462-1469], Marcelo, Filipa [0000-0001-5049-8511], Murphy, Paul V. [0000-0002-1529-6540], Birrane, Gabriel [0000-0002-1759-5499], UCIBIO - Applied Molecular Biosciences Unit, and DQ - Departamento de Química
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Glycan ,Acetylgalactosamine ,Tn antigen ,Crystallography, X-Ray ,Ligands ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Mice ,03 medical and health sciences ,Protein Domains ,SDG 3 - Good Health and Well-being ,C-type lectin ,Animals ,Humans ,Antigens, Tumor-Associated, Carbohydrate ,Lectins, C-Type ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Lectin ,Ligand (biochemistry) ,Transmembrane protein ,0104 chemical sciences ,3. Good health ,biology.protein ,Asialoglycoprotein receptor ,Glycoprotein ,Protein Binding - Abstract
10 p.-5 fig.-1 graph. abst., The human macrophage galactose lectin (MGL) is anendocytic type II transmembrane receptor expressed on immaturemonocyte-derived dendritic cells and activated macrophages and playsa role in modulating the immune system in response to infections andcancer. MGL contains an extracellular calcium-dependent (C-type)carbohydrate recognition domain (CRD) that specifically bindsterminalN-acetylgalactosamine glycan residues such as the Tn andsialyl-Tn antigens found on tumor cells, as well as otherN-andO-glycans displayed on certain viruses and parasites. Even though theglycan specificity of MGL is known and several binding glycoproteinshave been identified, the molecular basis for substrate recognition hasremained elusive due to the lack of high-resolution structures. Here wepresent crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactionswith the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like theasialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. Thestructure provides the molecular basis for preferential binding ofN-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance dataacquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution.Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisXand GalNAc,respectively, This work was supported by funding from Gabrielle’s Angel Foundation for Cancer Research to G.B., grants 12/IA/1398and 16/IA/4419 from Science Foundation Ireland to P.V.M.and GOIPG/2016/858 from the Irish Research Council toA.G. F.M., H.C., and A.D. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding ProjectsIF/00780/2015 and PTDC/BIA-MIB/31028/2017 and UCI-BIO Project UIDB/04378/2020, as well as the Ph.D. grantattributed to A.D. (PD/BD/142847/2018). The NMRspectrometers are part of the National NMR Network(PTNMR) and are partially supported by InfrastructureProject 22161 (co-financed by FEDER through COMPETE2020, POCI, and PORL and FCT through PIDDAC). F.J.C.acknowledges funding from Agencia Estatal de Investigación(Spain) for Grant RTI2018-094751-B-C22 and CIBERES, aninitiative from the Spanish Institute of Health Carlos III. F.C.thanks Agencia Estatal de Investigación (Spain) for Grant RTI2018-099592-B-C2.
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- 2021
3. Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen
- Author
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Gabba, Adele, primary, Bogucka, Agnieszka, additional, Luz, John G., additional, Diniz, Ana, additional, Coelho, Helena, additional, Corzana, Francisco, additional, Cañada, Francisco Javier, additional, Marcelo, Filipa, additional, Murphy, Paul V., additional, and Birrane, Gabriel, additional
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- 2021
- Full Text
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4. Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases
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Stephen Bell, Luet-Lok Wong, M.N. Podgorski, Jeanette E. Stok, Paul V. Bernhardt, Jeffrey Harmer, Jake A. Yorke, Joshua S. Harbort, T. Coleman, John B. Bruning, and James J. De Voss
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Models, Molecular ,Stereochemistry ,Heme ,010402 general chemistry ,Ligands ,01 natural sciences ,Biochemistry ,Benzoates ,Mixed Function Oxygenases ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Cytochrome P-450 Enzyme System ,030304 developmental biology ,Benzoic acid ,0303 health sciences ,Binding Sites ,biology ,Active site ,Cytochrome P450 ,Monooxygenase ,Ligand (biochemistry) ,0104 chemical sciences ,Kinetics ,Rhodopseudomonas ,chemistry ,Catalytic cycle ,biology.protein ,Protein Binding - Abstract
The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, β-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.
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- 2020
5. Kinetic and structural evidence for the importance of Tyr236 for the integrity of the Mo active site in a bacterial sulfite dehydrogenase
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&Kappler, Ulrike, &Bailey, Susan, &Feng, Changjian, &Honeychurch, Michael J., &Hanson, Graeme R., &Bernhardt, Paul V., &Tollin, Gordon, and &Enemark, John H.
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Electrochemistry -- Research ,Arginine -- Chemical properties ,Arginine -- Electric properties ,Arginine -- Spectra ,Electron transport -- Research ,Biological sciences ,Chemistry - Abstract
[SDH.sup.Y236F] is described in terms of its constitution, enzyme activity, electrochemically driven catalysis, intramolecular electron transfer and spectroscopic properties. As a result of substitution, the hydrogen bonding network adjoining the active site is disturbed, resulting in an increased mobility of nearby arginine.
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- 2006
6. Site-directed mutagenesis of dimethyl sulfoxide reductase from Rhodobacter capsulatus: characterization of a Y114 (approaches) F mutant
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Ridge, Justin P., Aguey-Zinsou, Kondo-Francois, Bernhardt, Paul V., Brereton, Ian M., Hanson, Graeme R., and McEwan, Alastair G.
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Biochemistry -- Research ,Gene expression -- Physiological aspects ,Mutagenesis -- Analysis ,Dimethyl sulfoxide -- Physiological aspects ,Gene mutations -- Analysis ,Biological sciences ,Chemistry - Abstract
Research has been conducted on dimethyl sulfoxide reductase from Rhodobacter capsulatus. The construction of the system used for expression site-directed mutants of this reductase in the natural host in order to generate and express dimethyl sulfoxide reductase with Y114F mutantion is described.
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- 2002
7. Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1
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Branson, Joy P., Nezic, Mark, Wallace, John C., and Attwood, Paul V.
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Biochemistry -- Research ,Brewer's yeast -- Genetic aspects ,Isoenzymes -- Genetic aspects ,Biotin -- Physiological aspects ,Carboxylic acids -- Physiological aspects ,Biological sciences ,Chemistry - Abstract
Research has been conducted on isoenzymes Pyc1 and Pyc2 from Saccharomyces cerevisiae the expression of which is differentially regulated. The kinetic characterization of Pyc1 has been carried out and the effects of acetyl CoA on the enzyme's kinetics have been investigated and the results are reported.
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- 2002
8. Mammalian histidine kinases: do the really exist?
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Tan, Eiling, Besant, Paul G., and Attwood, Paul V.
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Biochemistry -- Research ,Histidine -- Physiological aspects ,Protein kinases -- Physiological aspects ,Cells -- Physiological aspects ,Phosphorylation -- Physiological aspects ,Biological sciences ,Chemistry - Abstract
Research has been conducted on protein histidine phosphorylation in mammalian cells. The topics of interest include histidine kinases which are similar to mammalian protein kinases and possible biological roles of histidine kinases.
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- 2002
9. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase
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Liao, Der-Ing, Zheng, Ya-Jun, Viitanen, Paul V., and Jordan, Douglas B.
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Biochemistry -- Research ,Phosphates -- Physiological aspects ,Enzymes -- Physiological aspects ,Ligands -- Physiological aspects ,Biological sciences ,Chemistry - Abstract
Research has been conducted on the L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea. The X-ray crystal structures of this synthase has been examined and the high-resolution structures of the enzyme complexed with ligands are presented.
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- 2002
10. Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases
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Podgorski, Matthew N., primary, Harbort, Joshua S., additional, Coleman, Tom, additional, Stok, Jeanette E., additional, Yorke, Jake A., additional, Wong, Luet-Lok, additional, Bruning, John B., additional, Bernhardt, Paul V., additional, De Voss, James J., additional, Harmer, Jeffrey R., additional, and Bell, Stephen G., additional
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- 2020
- Full Text
- View/download PDF
11. Effects of Mg(super.2+) on the pre-steady state kinetics of the biotin carboxylation reaction of pyruvate carboxylase
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Branson, Joy P. and Attwood, Paul V.
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Magnesium -- Physiological aspects ,Biotin -- Physiological aspects ,Adenosine triphosphate -- Physiological aspects ,Pyrvinium -- Physiological aspects ,Carboxylic acids -- Physiological aspects ,Chemical reaction, Rate of -- Physiological aspects ,Biological sciences ,Chemistry - Abstract
Mg(super.2+) concentration and its effects on the pre-steady state kinetics of the biotin carboxylation reaction of pyruvate carboxylase have been studied. The enzyme was found to undergo dilution inactivation at low Mg(super.2+) concentrations. This happened at higher enzyme concentrations than had been seen before. The major foci of action of Mg(super.2+) appear to be at the decarboxylation of the enzyme-carboxybiotin complex, the return of the biotin to the site of the biotin carboxylation reaction, and the coupling of ATP cleavage to biotin carboxylation.
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- 2000
12. Effects of acetyl coA on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase
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Legge, Glen B., Branson, Joy P., and Attwood, Paul V.
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Enzymes -- Synthesis ,Biotin -- Research ,Biological sciences ,Chemistry - Abstract
First-order kinetics determines the access to the steady-state for the synthesis of the enzyme-carboxybiotin complex. The rate constants with or without acetyl CoA have been identified as 6.6 and 0.028 s-1 for ATP to cleave and 6.1 and 0.028 s-10 to form enzyme-carboxybiotin complexes. Acetyl CoA magnified the rate constants for the approach to steady-state kinetics for ATP cleavage and carboxyenzyme synthesis. The lack of acetyl CoA lead to the fast disintegration of carboxyphosphate.
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- 1996
13. Kinetics of nucleotide binding to pyruvate carboxylase
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Geeves, Michael A., Branson, Joy P., and Attwood, Paul V.
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Nucleotides -- Analysis ,Protein binding -- Analysis ,Dynamics -- Research ,Biological sciences ,Chemistry - Abstract
Kinetics of nucleotide binding to pyruvate carboxylase is measured by noting the fluorescence changes that occurred on the formycin nucleotide binding to the enzymes using the stopped-flow process. The effects of the cofactors Mg2+ and acetyl coenzyme a on these kinetics, and the effects of pyruvate and bicarbonate ion on nucleotide binding are determined. The rate constants of the slow component of Mg*formycin nucleotide binding is independent of nucleotide concentration.
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- 1995
14. Locus of action of acetyl CoA in the biotin-carboxylation reaction of pyruvate carboxylase
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Attwood, Paul V.
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Glucose metabolism -- Analysis ,Enzymes -- Analysis ,Biological sciences ,Chemistry - Abstract
An analysis of pyruvate carboxylase's biotin-carboxylation reaction reveals that the only major acetyl CoA locus of action in pyruvate carboxylase activation is the increase in HCO3-dependent ATP-breakup reaction. Acetyl CoA is unnecessary for carboxyl group transfer from the carboxyphospho-enzyme (CPE) complex to pyruvate. The lack of reactivity loss of CPE on reaction with MgADP implies that CPE is not the required enzyme-carboxyphosphate complex.
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- 1993
15. Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion
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Todd, Matthew J., Viitanen, Paul V., and Lorimer, George H.
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Adenosine triphosphate -- Research ,Potassium channels -- Physiological aspects ,Escherichia coli -- Research ,Biological sciences ,Chemistry - Abstract
The ATP concentration inversely depends upon the potassium-ion activation constant (K-act) for the ATPase activity of Escherichia coli chaperonin groEL. Positive cooperative kinetics is caused by the APase activity. Increased concentration of potassium leads to a decrease in the K0.5 of ATP. The absence of potassium causes MgATP to bind weakly to groEl when hydrolysis does not take place. Potassium-dependent hydrolysis of ATP is unaffected by the absence of groEL, while the presence of groES makes the process complicated. The repressed state can be recreated when the potassium level is low and free-MgADP is absent.
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- 1993
16. Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA
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Chaiyos Sirithanakorn, Sarawut Jitrapakdee, and Paul V. Attwood
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Stereochemistry ,Allosteric regulation ,Glutamic Acid ,Propionyl-CoA carboxylase ,Cooperativity ,Arginine ,Rhizobium etli ,Biochemistry ,Serine ,03 medical and health sciences ,chemistry.chemical_compound ,Allosteric Regulation ,Bacterial Proteins ,Acetyl Coenzyme A ,Methylcrotonyl-CoA carboxylase ,Amino Acid Sequence ,Pyruvate Carboxylase ,Aspartic Acid ,030102 biochemistry & molecular biology ,Acetyl-CoA ,Acetyl-CoA binding ,Recombinant Proteins ,Pyruvate carboxylase ,Enzyme Activation ,Kinetics ,chemistry ,Mutagenesis, Site-Directed ,Allosteric Site - Abstract
The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis.
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- 2016
17. Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60
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Vies, Saskia M. van der, Viitanen, Paul V., Gatenby, Anthony A., Lorimer, George H., and Jaenicke, Rainer
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Ribulosebiphosphate carboxylase -- Research ,Enzymes ,Proteins -- Conformation ,Biological sciences ,Chemistry - Abstract
Conformational analysis of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum revealed two acid-denatured states and two folded states. The acid-denatured states were comprised of UA1 state, an unfolded and monomeric conformation at pH2 and low ionic strength and A1 state, which was an intermediate conformation at the same pH but at high ionic strength. N1 and N2 states comprised the folded conformational states and were noted be homologous in their secondary state. An unstable fifth conformational state was observed and was found to form a stable binary complex in the presence of chaperonin 60 oligomer.
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- 1992
18. Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by <scp>l</scp>-Aspartate
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Abdussalam Adina-Zada, Sarawut Jitrapakdee, John C. Wallace, Paul V. Attwood, and Chaiyos Sirithanakorn
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Pyruvate decarboxylation ,Aspartic Acid ,Pyruvate dehydrogenase kinase ,endocrine system diseases ,biology ,Chemistry ,nutritional and metabolic diseases ,Pyruvate dehydrogenase phosphatase ,Rhizobium etli ,Biochemistry ,Article ,Pyruvate carboxylase ,Citric acid cycle ,Non-competitive inhibition ,biology.protein ,Enzyme Inhibitors ,Enzyme inducer ,hormones, hormone substitutes, and hormone antagonists ,Pyruvate Carboxylase - Abstract
l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. l-Aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, l-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and l-aspartate) are spatially distinct but connected by a network of allosteric interactions.
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- 2014
19. Probing the catalytic roles of Arg548 and Gln552 in the carboxyl transferase domain of the Rhizobium etli pyruvate carboxylase by site-directed mutagenesis
- Author
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Duangpan, Saowapa, Jitrapakdee, Sarawut, Adina-Zada, Abdussalam, Zeczycki, Tonya N., Byrne, Lindsay, Cleland, W. Wallace, Maurice, Martin St., Attwood, Paul V., and Wallace, John C.
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Biotin -- Chemical properties ,Biotin -- Structure ,Enzyme binding -- Analysis ,Mutagenesis -- Analysis ,Pyruvate carboxylase -- Structure ,Pyruvate carboxylase -- Chemical properties ,Rhizobium -- Physiological aspects ,Transferases -- Chemical properties ,Biological sciences ,Chemistry - Abstract
Site-directed mutagenesis was used to study the roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase. The results suggested that Arg548 and Gln552 facilitated the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase.
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- 2010
20. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family
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Harris, Paul V., Welner, Ditte, McFarland, K.C., Re, Edward, Poulsen, Jens-Christian Navarro, Brown, Kimberly, Salbo, Rune, Hanshu Ding, Vlasenko, Elena, Merino, Sandy, Feng Xu, Cherry, Joel, Larsen, Sine, and Leggio, Leila Lo
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Catalysis -- Analysis ,Cellulose -- Chemical properties ,Alcohol -- Chemical properties ,Alcohol, Denatured -- Chemical properties ,Glycosides -- Chemical properties ,Hydrolases -- Chemical properties ,Enzymes -- Chemical properties ,Biological sciences ,Chemistry - Abstract
The structural and functional studies on the enigmatic family of glycoside hydrolase 61 (GH61) that catalyze cellulose and hemicellulose hydrolysis are presented. The total protein loading required to hydrolyze lignocellulosic biomass could be reduced to 2-fold by incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes.
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- 2010
21. Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from
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Rhizobium etli, Zeczycki, Tonya N., Maurice, Martin St., Jitrapakdee, Sarawut, Wallace, John C., Attwood, Paul V., and Cleland, W. Wallace
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Biotin -- Chemical properties ,Carboxylation -- Analysis ,Mutation (Biology) -- Analysis ,Rhizobium -- Physiological aspects ,Rhizobium -- Genetic aspects ,Transferases -- Chemical properties ,Biological sciences ,Chemistry - Abstract
Mutagenic studies were conducted to examine the effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase. The results provide insight into the putative roles of catalytically relevant active site residues and the mechanism of the carboxyl transferase domain which involved the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882.
- Published
- 2009
22. Intramolecular electron transfer in sulfite-oxidizing enzymes: elucidating the role of a conserved active site arginine
- Author
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Emesh, Safia, Rapson, Trevor D., Rajapakshe, Asha, Kappler, Ulrike, Bernhardt, Paul V., Tollin, Gordon, and Enemark, John H.
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Arginine -- Chemical properties ,Electron transport -- Analysis ,Oxidation-reduction reaction -- Analysis ,Oxidoreductases -- Chemical properties ,Oxidoreductases -- Structure ,Sulfides -- Chemical properties ,Biological sciences ,Chemistry - Abstract
The equivalent uncharged substitutions R55Q and R55M as well as the positively charged substitution R55K in bacterial sulfite dehydrogenase (SDH) were explored to understand their role towards intramolecular electron transfer (IET). The results provide insight into the inhibition of [SDH.sup.R55Q] variant in IET by sulfate and possible mechanistic pathways for sulfide-oxidizing enzymes.
- Published
- 2009
23. Roles of Arg427 and Arg472 in the Binding and Allosteric Effects of Acetyl CoA in Pyruvate Carboxylase
- Author
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Chutima Sereeruk, Tonya N. Zeczycki, W. Wallace Cleland, Martin St. Maurice, Sarawut Jitrapakdee, Abdussalam Adina-Zada, John C. Wallace, and Paul V. Attwood
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Models, Molecular ,Biotin carboxylase ,Allosteric regulation ,Acetyl-CoA ,Biotin ,Propionyl-CoA carboxylase ,Biology ,Arginine ,Biochemistry ,Molecular biology ,Article ,Pyruvate carboxylase ,Adenosine Diphosphate ,chemistry.chemical_compound ,Adenosine Triphosphate ,Allosteric Regulation ,chemistry ,Acetyl Coenzyme A ,Methylcrotonyl-CoA carboxylase ,Carbamoyl phosphate ,Mutagenesis, Site-Directed ,Phosphorylation ,Pyruvate Carboxylase - Abstract
Mutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation constant (K(a)) of acetyl CoA, with the increase being greater for the Arg472 mutants. These results indicate that while both these residues are involved in the binding of acetyl CoA to the enzyme, Arg472 is more important than Arg427. The mutations had substantially smaller effects on the k(cat) for pyruvate carboxylation. Part of the effects of the mutations was to increase the K(m) for MgATP and the K(a) for activation by free Mg(2+) determined at saturating acetyl CoA concentrations. The inhibitory effects of the mutations on the rates of the enzyme-catalyzed bicarbonate-dependent ATP cleavage, carboxylation of biotin, and phosphorylation of ADP by carbamoyl phosphate indicate that the major locus of the effects of the mutations was in the biotin carboxylase (BC) domain active site. Even though both Arg427 and Arg472 are distant from the BC domain active site, it is proposed that their contacts with other residues in the allosteric domain, either directly or through acetyl CoA, affect the positioning and orientation of the biotin-carboxyl carrier protein (BCCP) domain and thus the binding of biotin at the BC domain active site. On the basis of the kinetic analysis proposed here, it is proposed that mutations of Arg427 and Arg472 perturb these contacts and consequently the binding of biotin at the BC domain active site. Inhibition of pyruvate carboxylation by the allosteric inhibitor l-aspartate was largely unaffected by the mutation of either Arg427 or Arg472.
- Published
- 2012
24. Thermodynamic characterization of the redox centers within dimethysulfide dehydrogenase
- Author
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Creevey, Nicole L., McEwan, Alastair G., Hanson, Graeme R., and Bernhardt, Paul V.
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Oxidation-reduction reaction -- Analysis ,Oxidoreductases -- Chemical properties ,Oxidoreductases -- Thermal properties ,Bacteria, Photosynthetic -- Environmental aspects ,Bacteria, Photosynthetic -- Composition ,Redox potential -- Evaluation ,Biological sciences ,Chemistry - Abstract
The thermodynamic characterization of the redox centers within dimethylsulfide (DMS) dehydrogenase, a complex heterotrimeric enzyme that catalyzes the oxidation of DMS to DMSO in Rhodovulum sulfidophilum is described. The results from comparison to other bacterial Mo enzymes such as NarGHI nitrate reductase, selenate reductase and ethylbenzene dehydrogenase indicate remarkable similarity in the redox potential of all Fe-S clusters.
- Published
- 2008
25. Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli
- Author
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Sarawut Jitrapakdee, John C. Wallace, Tonya N. Zeczycki, W. Wallace Cleland, Paul V. Attwood, Martin St. Maurice, and Ann L. Menefee
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Oxaloacetic Acid ,Phosphonoacetic Acid ,inorganic chemicals ,Biotin carboxylase ,Rhizobium etli ,Biochemistry ,Article ,chemistry.chemical_compound ,Adenosine Triphosphate ,Bacterial Proteins ,Biotin ,Acetyl Coenzyme A ,Catalytic Domain ,Oxaloacetic acid ,Transferase ,Magnesium ,Enzyme Inhibitors ,Phosphorylation ,Pyruvate Carboxylase ,Hydrogen-Ion Concentration ,Recombinant Proteins ,Protein Structure, Tertiary ,Pyruvate carboxylase ,Adenosine Diphosphate ,Enzyme Activation ,Kinetics ,chemistry ,Carboxylation ,Mutagenesis, Site-Directed ,Adenosine triphosphate - Abstract
While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.
- Published
- 2011
26. Novel Insights into the Biotin Carboxylase Domain Reactions of Pyruvate Carboxylase from Rhizobium etli
- Author
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John C. Wallace, Kathy H. Surinya, Paul V. Attwood, Tonya N. Zeczycki, Abdussalam Adina-Zada, Sarawut Jitrapakdee, Martin St. Maurice, Ann L. Menefee, and W. Wallace Cleland
- Subjects
Models, Molecular ,Oxaloacetic Acid ,Biotin carboxylase ,Stereochemistry ,Biotin ,Rhizobium etli ,Biochemistry ,Article ,chemistry.chemical_compound ,Adenosine Triphosphate ,Bacterial Proteins ,Catalytic Domain ,Oxaloacetic acid ,Carbon-Nitrogen Ligases ,Enzyme kinetics ,Protein Structure, Quaternary ,Pyruvate Carboxylase ,biology ,Active site ,Recombinant Proteins ,Protein Structure, Tertiary ,Pyruvate carboxylase ,Kinetics ,chemistry ,Carboxylation ,Mutagenesis, Site-Directed ,biology.protein - Abstract
The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO(3)(-), Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in k(cat) for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO(3)(-) by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO(2) and PO(4)(3-). PO(4)(3-) then acts as the base to deprotonate the tethered biotin at the N(1)-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO(2) to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain.
- Published
- 2011
27. A photoaffinity analogue discodermolide specifically labels a peptide in [beta]-tubulin
- Author
-
Shujun Xia, Kenesky, Craig S., Rucker, Paul V., Smith, Amos B., III, Orr, George A., and Horwitz, Susan Band
- Subjects
Affinity labeling -- Research ,Peptides -- Atomic properties ,Nucleation -- Research ,Biological sciences ,Chemistry - Abstract
Photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity.
- Published
- 2006
28. Protein film voltammetry of arsenite oxidase from the chemolithoautotrophic arsenite-oxidizing bacterium NT-26
- Author
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Bernhardt, Paul V. and Santini, Joanne M.
- Subjects
Voltammetry -- Analysis ,Arsenic -- Environmental aspects ,Arsenic -- Chemical properties ,Electrochemistry -- Research ,Biological sciences ,Chemistry - Abstract
A study of the protein film voltammetry of arsenite oxidase from the chemolithoautotrophic arsenite-oxidizing bacterium is presented. The enzyme functions natively while absorbed on the electrode where it displays stable and reproducible catalytic electrochemistry in the presence of arsenite.
- Published
- 2006
29. Insight into the Carboxyl Transferase Domain Mechanism of Pyruvate Carboxylase from Rhizobium etli
- Author
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Sarawut Jitrapakdee, W. Wallace Cleland, John C. Wallace, Tonya N. Zeczycki, Paul V. Attwood, and Martin St. Maurice
- Subjects
Models, Molecular ,Threonine ,Biotin carboxylase ,Carboxy-lyases ,Oxaloacetates ,Stereochemistry ,Decarboxylation ,Molecular Conformation ,Rhizobium etli ,Biochemistry ,Article ,chemistry.chemical_compound ,Biotin ,Oxaloacetic acid ,Escherichia coli ,Transferase ,Phosphorylation ,Pyruvates ,Pyruvate Carboxylase ,Adenosine Triphosphatases ,biology ,Chemistry ,Active site ,Protein Structure, Tertiary ,Pyruvate carboxylase ,Kinetics ,Carboxyl and Carbamoyl Transferases ,biology.protein ,Protons ,Allosteric Site - Abstract
The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO(2) to form oxaloacetate and complete the reaction.
- Published
- 2009
30. Thermodynamic Characterization of the Redox Centers within Dimethylsulfide Dehydrogenase
- Author
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Nicole L. Creevey, Alastair G. McEwan, Paul V. Bernhardt, and Graeme R. Hanson
- Subjects
Iron-Sulfur Proteins ,Molybdenum ,Binding Sites ,Rhodovulum ,biology ,Chemistry ,Stereochemistry ,fungi ,Dehydrogenase ,Electron donor ,Nitrate reductase ,biology.organism_classification ,Biochemistry ,Redox ,Cofactor ,Selenate reductase ,chemistry.chemical_compound ,biology.protein ,Thermodynamics ,Oxidoreductases ,Oxidation-Reduction ,Heme - Abstract
Dimethylsulfide (DMS) dehydrogenase is a complex heterotrimeric enzyme that catalyzes the oxidation of DMS to DMSO and allows Rhodovulum sulfidophilum to grow under photolithotrophic conditions with DMS as the electron donor. The enzyme is a 164 kDa heterotrimer composed of an alpha-subunit that binds a bis(molybdopterin guanine dinucleotide)Mo cofactor, a polyferredoxin beta-subunit, and a gamma-subunit that contains a b-type heme. In this study, we describe the thermodynamic characterization of the redox centers within DMS dehydrogenase using EPR- and UV-visible-monitored potentiometry. Our results are compared with those of other bacterial Mo enzymes such as NarGHI nitrate reductase, selenate reductase, and ethylbenzene dehydrogenase. A remarkable similarity in the redox potentials of all Fe-S clusters is apparent.
- Published
- 2008
31. Direct electrochemistry of porcine purple acid phosphate (Uteroferrin)
- Author
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Bernhardt, Paul V., Schenk, Gerhard, and Wilson, Gregory J.
- Subjects
Superphosphates -- Composition ,Proteins -- Chemical properties ,Proteins -- Research ,Chemistry, Analytic ,Biological sciences ,Chemistry - Abstract
The purple acid phosphates (PAPs) comprise a subfamily of non-heme dinuclear iron containing proteins that also include the ribonucleotide reductases, methane monooxygenases and hemerythrins. The first voltammetric investigation of an interesting class of enzymes and the effect of other inhibitors on the redox properties of these proteins are represented.
- Published
- 2004
32. Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A
- Author
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Branson, Joy P., Nezic, Mark, Jitrapakdee, Sarawut, Wallace, John C., and Attwood, Paul V.
- Subjects
Biochemistry -- Research ,Pyruvates -- Properties ,Pyruvates -- Research ,Enzymatic analysis ,Biological sciences ,Chemistry - Abstract
The yeast Pyc1 (pyruvate carboxylase) isoform of pyruvate carboxylase is characterized and shown to differ from the Pyc2 isoform in its K (sub a) for K (super +) activation. Kinetic measurements on the C249A mutant enzyme reveals that C249 is involved in the binding and action of enzyme activators K (super +) and acetyl-CoA.
- Published
- 2004
33. Kinetic Characterization of Yeast Pyruvate Carboxylase Isozyme Pyc1 and the Pyc1 Mutant, C249A
- Author
-
Mark Nezic, Sarawut Jitrapakdee, Joy P. Branson, Paul V. Attwood, and John C. Wallace
- Subjects
Pyruvate decarboxylation ,Biotin carboxylase ,Carbamyl Phosphate ,Saccharomyces cerevisiae Proteins ,Pyruvate dehydrogenase kinase ,Molecular Sequence Data ,Pyruvate dehydrogenase phosphatase ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Acetyl Coenzyme A ,Carbamoyl phosphate ,Carbon-Nitrogen Ligases ,Amino Acid Sequence ,Cysteine ,Phosphorylation ,Pyruvate Carboxylase ,Alanine ,Chemistry ,Escherichia coli Proteins ,Deuterium Exchange Measurement ,Carbamoyl phosphate synthetase ,Pyruvate dehydrogenase complex ,Pyruvate carboxylase ,Adenosine Diphosphate ,Isoenzymes ,Bicarbonates ,Kinetics ,Mutagenesis, Site-Directed ,Solvents ,o-Phthalaldehyde ,Acetyl-CoA Carboxylase - Abstract
The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.
- Published
- 2003
34. Site-Directed Mutagenesis of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus: Characterization of a Y114 → F Mutant
- Author
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Alastair G. McEwan, Graeme R. Hanson, Ian M. Brereton, Paul V. Bernhardt, Justin P. Ridge, and Kondo-Francois Aguey-Zinsou
- Subjects
Iron-Sulfur Proteins ,Phenylalanine ,Mutant ,Biochemistry ,Rhodobacter capsulatus ,chemistry.chemical_compound ,Bacterial Proteins ,Oxidoreductase ,Electrochemistry ,Dimethyl Sulfoxide ,Enzyme kinetics ,Molybdenum ,chemistry.chemical_classification ,DMSO reductase ,Binding Sites ,Rhodobacter ,biology ,Dimethyl sulfoxide ,Molybdopterin ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Kinetics ,Amino Acid Substitution ,chemistry ,Mutagenesis, Site-Directed ,Tyrosine ,Spectrophotometry, Ultraviolet ,Dimethyl sulfide ,Oxidoreductases ,Oxidation-Reduction ,Protein Binding - Abstract
A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.
- Published
- 2002
35. Kinetic Characterization of Yeast Pyruvate Carboxylase Isozyme Pyc1
- Author
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Mark Nezic, Joy P. Branson, Paul V. Attwood, and John C. Wallace
- Subjects
Pyruvate decarboxylation ,Saccharomyces cerevisiae Proteins ,Time Factors ,Pyruvate dehydrogenase kinase ,Biotin ,Saccharomyces cerevisiae ,PKM2 ,Pyruvate dehydrogenase phosphatase ,Biology ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Adenosine Triphosphate ,Acetyl Coenzyme A ,Methylcrotonyl-CoA carboxylase ,Protein Isoforms ,Pyruvate Carboxylase ,Acetyl-CoA ,Temperature ,Pyruvate dehydrogenase complex ,Molecular biology ,Pyruvate carboxylase ,Kinetics ,Models, Chemical ,chemistry ,Protein Binding - Abstract
Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for pyruvate carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast pyruvate carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the K(a) of this isozyme for acetyl CoA is in the order of 8-10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver pyruvate carboxylase. However, unlike the chicken liver enzyme, the quaternary structure of the enzyme is quite stable in the absence of acetyl CoA, and the major locus of action of this effector appears to lie outside of the stimulation of the biotin carboxylation reaction.
- Published
- 2002
36. Mammalian Histidine Kinases: Do They REALLY Exist?
- Author
-
Eiling Tan, Paul V. Attwood, and Paul G. Besant
- Subjects
Mammals ,Histidine Kinase ,biology ,Kinase ,Chemistry ,Histidine kinase ,Biochemistry ,Histones ,Histone ,biology.protein ,Animals ,Phosphorylation ,Protein Kinases ,Histidine - Published
- 2002
37. Structural Definition of the Active Site and Catalytic Mechanism of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase
- Author
-
Paul V. Viitanen, Der-Ing Liao, Ya-Jun Zheng, and Douglas B. Jordan
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Stereoisomerism ,Crystal structure ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Catalytic Domain ,Side chain ,Imidazole ,Intramolecular Transferases ,biology ,Chemistry ,Hydrogen bond ,Active site ,Substrate (chemistry) ,Hydrogen Bonding ,Recombinant Proteins ,Enzyme Activation ,Magnaporthe ,Models, Chemical ,Catalytic cycle ,biology.protein ,Thermodynamics - Abstract
X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The modelmore » suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.« less
- Published
- 2002
38. Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA
- Author
-
Sirithanakorn, Chaiyos, primary, Jitrapakdee, Sarawut, additional, and Attwood, Paul V., additional
- Published
- 2016
- Full Text
- View/download PDF
39. Effects of Acetyl CoA on the Pre-Steady-State Kinetics of the Biotin Carboxylation Reaction of Pyruvate Carboxylase
- Author
-
Joy P. Branson, Paul V. Attwood, and Glen B. Legge
- Subjects
Decarboxylation ,Stereochemistry ,Acetyl-CoA ,Biotin ,Propionyl-CoA carboxylase ,Biochemistry ,Phosphates ,Pyruvate carboxylase ,Kinetics ,chemistry.chemical_compound ,Adenosine Triphosphate ,Organophosphorus Compounds ,Liver ,Models, Chemical ,chemistry ,Carboxylation ,Acetyl Coenzyme A ,Methylcrotonyl-CoA carboxylase ,Animals ,Regression Analysis ,Steady state (chemistry) ,Chickens ,Pyruvate Carboxylase - Abstract
The effects of Mg(2+) concentration on the kinetics of both ATP cleavage and carboxyenzyme formation in the approach to steady state of the biotin carboxylation reaction of pyruvate carboxylase have been studied. It was found that the enzyme underwent dilution inactivation at low Mg(2+) concentrations and that this occurred at higher enzyme concentrations than had been previously observed. At 10 mM Mg(2+), dilution inactivation was prevented and activation of the enzyme also occurred. When the enzyme was mixed with an ATP solution to initiate the carboxylation reaction, dilution inactivation was reversed and further enzyme activation was induced to a final level that was dependent on Mg(2+) concentration. With the exception of the reaction at 10 mM Mg(2+) in the presence of acetyl CoA, the experimental data could be adequately described as first-order exponential approaches to steady state. At 10 mM Mg(2+) in the presence of acetyl CoA, both ATP cleavage and carboxyenzyme formation data were best described as a biexponential process, in which there was little ATP turnover at steady state. Modeling studies have been performed which produced simulated data that were similar to the experimental data, using a reaction scheme modified from one proposed previously [Legge, G. B., et al. (1996) Biochemistry 35, 3849-3856]. These studies indicate that the major foci of action of Mg(2+) are in the decarboxylation of the enzyme-carboxybiotin complex, the return of the biotin to the site of the biotin carboxylation reaction, and the coupling of ATP cleavage to biotin carboxylation.
- Published
- 1996
40. Kinetics of Nucleotide Binding to Pyruvate Carboxylase
- Author
-
Paul V. Attwood, Joy P. Branson, and Michael A. Geeves
- Subjects
Pyruvate decarboxylation ,Pyruvate dehydrogenase kinase ,In Vitro Techniques ,Pyruvate dehydrogenase phosphatase ,Binding, Competitive ,Models, Biological ,Biochemistry ,chemistry.chemical_compound ,Adenosine Triphosphate ,Acetyl Coenzyme A ,Pyruvic Acid ,Animals ,Magnesium ,Nucleotide ,Dihydrolipoyl transacetylase ,Pyruvates ,Fluorescent Dyes ,Pyruvate Carboxylase ,chemistry.chemical_classification ,Binding Sites ,Acetyl-CoA ,Ribonucleotides ,Pyruvate dehydrogenase complex ,Pyruvate carboxylase ,Bicarbonates ,Kinetics ,Spectrometry, Fluorescence ,Liver ,chemistry ,Biophysics ,Chickens ,Formycins - Abstract
The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1995
41. Interaction between the biotin carboxyl carrier domain and the biotin carboxylase domain in pyruvate carboxylase from Rhizobium etli
- Author
-
John C. Wallace, Martin St. Maurice, A.D. Lietzan, Ann L. Menefee, Sudhanshu Kumar, Paul V. Attwood, W. Wallace Cleland, and Tonya N. Zeczycki
- Subjects
Biotin carboxylase ,Models, Molecular ,Phosphonoacetic Acid ,Staphylococcus aureus ,Biotin carboxyl carrier protein ,Biotin ,Crystallography, X-Ray ,Biochemistry ,Rhizobium etli ,Article ,chemistry.chemical_compound ,Bacterial Proteins ,Species Specificity ,Methylcrotonyl-CoA carboxylase ,Catalytic Domain ,Transferase ,Carbon-Nitrogen Ligases ,Protein Structure, Quaternary ,DNA Primers ,Pyruvate Carboxylase ,biology ,Base Sequence ,Active site ,Recombinant Proteins ,Pyruvate carboxylase ,Protein Structure, Tertiary ,Kinetics ,chemistry ,biology.protein ,Mutagenesis, Site-Directed - Abstract
Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 A resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family.
- Published
- 2011
42. Locus of action of acetyl CoA in the biotin-carboxylation reaction of pyruvate carboxylase
- Author
-
Paul V. Attwood
- Subjects
Reaction mechanism ,Oxaloacetates ,Decarboxylation ,Stereochemistry ,Carboxylic Acids ,Malates ,Biotin ,Biochemistry ,chemistry.chemical_compound ,Acetyl Coenzyme A ,Animals ,Magnesium ,Pyruvates ,Incubation ,Edetic Acid ,Pyruvate Carboxylase ,chemistry.chemical_classification ,Acetyl-CoA ,Avidin ,Pyruvate carboxylase ,Adenosine Diphosphate ,Enzyme Activation ,Enzyme ,Liver ,chemistry ,Carboxylation ,Phosphotransferases (Phosphomutases) ,Chickens - Abstract
The [14C]carboxyphospho-enzyme complex formed by incubation of the enzyme with H14CO3-, MgATP, and Mg2+ was prepared and isolated by gel filtration as described by Phillips et al. [(1992) Biochemistry 31, 9445-9450]. When time courses of transfer of the [14C]carboxyl group from the complex to pyruvate were studied, it was found that at the first time point (15 s) the formation of [14C]oxalacetate was the same in the presence or absence of acetyl CoA. However, in the absence of acetyl CoA, the radioactivity fixed in [14C]oxalacetate declined rapidly over the subsequent 15 min, whereas in the presence of acetyl CoA the formation of [14C]oxalacetate continued up to about 10 min. The decline in [14C]oxalacetate in the absence of acetyl CoA was found to be due to enzyme-dependent decarboxylation of the oxalacetate by the enzyme. Incubation of the isolated [14C]carboxyphospho-enzyme complex with MgADP and Mg2+ resulted in no significant reduction in the formation of [14C]oxalacetate on addition of acetyl CoA and pyruvate. Incubation of the isolated [32P]carboxyphospho-enzyme complex with pyruvate resulted in no significant reduction in the formation of [gamma-32P]ATP on the addition of MgADP and Mg2+. This new evidence casts doubt on the suggested locus of activation of the enzyme by acetyl CoA being the facilitation of the transfer of the carboxyl group from carboxyphosphate to biotin and indeed on the identity of the isolated enzyme intermediate [Phillips et al. (1992) Biochemistry 31, 9445-9450].
- Published
- 1993
43. Mechanisms of Inhibition of Rhizobium etli Pyruvate Carboxylase by l-Aspartate
- Author
-
Sirithanakorn, Chaiyos, primary, Adina-Zada, Abdussalam, additional, Wallace, John C., additional, Jitrapakdee, Sarawut, additional, and Attwood, Paul V., additional
- Published
- 2014
- Full Text
- View/download PDF
44. Intramolecular electron transfer in sulfite-oxidizing enzymes: elucidating the role of a conserved active site arginine
- Author
-
John H. Enemark, S. Asha Rajapakshe, Paul V. Bernhardt, Safia Emesh, Ulrike Kappler, Gordon Tollin, and Trevor D. Rapson
- Subjects
Models, Molecular ,Cytochrome ,Arginine ,Stereochemistry ,Biochemistry ,Article ,Electron Transport ,chemistry.chemical_compound ,Sulfite ,Catalytic Domain ,Proteobacteria ,Humans ,Sulfite dehydrogenase ,Oxidoreductases Acting on Sulfur Group Donors ,Heme ,Sulfite oxidase deficiency ,Photolysis ,biology ,Chemistry ,Sulfates ,Lasers ,Active site ,Hydrogen-Ion Concentration ,Recombinant Proteins ,Kinetics ,Catalytic cycle ,Amino Acid Substitution ,Models, Chemical ,biology.protein ,Oxidation-Reduction - Abstract
All reported sulfite-oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. To improve our understanding of the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions R55Q and R55M as well as the positively charged substitution R55K in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for improving our understanding of residue function in IET without the added complexity of the interdomain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) exhibited increased IET rate constants relative to that of the wild type (3-4-fold) when studied by laser flash photolysis. The gain in function observed in SDH(R55Q) and SDH(R55M) suggests that the reduction in the level of IET seen in HSO(R160Q) is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the interdomain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDH(WT), SDH(R55M), and SDH(R55Q) show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (K(eq)) and rate constants (k(et)) of both variants with respect to the SDH(WT) enzyme. SDH(WT) has a k(et) that is independent of pH and a K(eq) that increases as pH decreases; on the other hand, both SDH(R55M) and SDH(R55Q) have a k(et) that increases as pH decreases, and SDH(R55M) has a K(eq) that is pH-independent. IET in the SDH(R55Q) variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from that of SDH(WT), but which also occurs in HSO. IET in SDH(R55K) is slower than in SDH(WT). A new analysis of the possible mechanistic pathways for sulfite-oxidizing enzymes is presented and related to available kinetic and EPR results for these enzymes.
- Published
- 2009
45. Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase
- Author
-
Paul V. Viitanen, Gail K. Donaldson, Anthony A. Gatenby, George H. Lorimer, and Thomas H. Lubben
- Subjects
GroES Protein ,Macromolecular Substances ,Protein Conformation ,Biochemistry ,Chaperonin ,Mice ,Adenosine Triphosphate ,Bacterial Proteins ,ATP hydrolysis ,Enzyme Stability ,Dihydrofolate reductase ,Escherichia coli ,Animals ,Heat-Shock Proteins ,biology ,Chaperonin 60 ,GroES ,GroEL ,Enzyme Activation ,Tetrahydrofolate Dehydrogenase ,enzymes and coenzymes (carbohydrates) ,Chaperone (protein) ,biology.protein ,Folic Acid Antagonists ,Protein folding - Abstract
The spontaneous refolding of chemically denatured dihydrofolate reductase (DHFR) is completely arrested by chaperonin 60 (GroEL). This inhibition presumably results from the formation of a stable complex between chaperonin 60 and one or more intermediates in the folding pathway. While sequestered on chaperonin 60, DHFR is considerably more sensitive to proteolysis, suggesting a nonnative structure. Bound DHFR can be released from chaperonin 60 with ATP, and although chaperonin 10 (GroES) is not obligatory, it does potentiate the maximum effect of ATP. Hydrolysis of ATP is also not required for DHFR release since certain nonhydrolyzable analogues are capable of partial discharge. "Native" DHFR can also form a stable complex with chaperonin 60. However, in this case, complex formation is not instantaneous and can be prevented by the presence of DHFR substrates. This suggests that native DHFR exists in equilibrium with at least one conformer which is recognizable by chaperonin 60. Binding studies with 3SS-labeled DHFR support these conclusions and further demonstrate that DHFR competes for a common saturable site with another protein (ribulose-l,5-bisphosphate carboxylase) known to interact with chaperonin 60. Numerous in vitro studies on the folding pathways of chemically denatured proteins have demonstrated that many proteins successfully achieve their correct native structures by using information contained in the primary amino acid se- quence (reviewed by Creighton (1990) and Jaenicke (1987)l. This has led to the general view that protein folding in vivo is also a spontaneous event. However, the cellular reality, for some proteins at least, may be quite different. In part this is due to a temporal element, whereby nascent polypeptides emerge from ribosomes in a vectorial fashion and are subject to the initiation of folding in the absence of the completed chain. A similar situation likely pertains to polypeptides which are translocated across biological membranes. To this must be added the chemical complexity of the cell, in which high concentrations of proteins in various states of folding, and with potentially interactive surfaces, must surely coexist. A class of proteins termed chaperonins (Hemmingsen et al., 1988) have been identified that affect the folding and subsequent assembly of proteins either
- Published
- 1991
46. Chaperonin-facilitated refolding of ribulose bisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are potassium dependent
- Author
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George H. Lorimer, Paul V. Viitanen, Janet E. Reed, Thomas H. Lubben, Pierre Goloubinoff, and Daniel P. O'Keefe
- Subjects
inorganic chemicals ,GroES Protein ,Chaperonins ,Protein Conformation ,Ribulose-Bisphosphate Carboxylase ,macromolecular substances ,Biochemistry ,Chaperonin ,Adenosine Triphosphate ,Bacterial Proteins ,ATP hydrolysis ,Escherichia coli ,GroEL Protein ,Adenosine Triphosphatases ,biology ,Chemistry ,Hydrolysis ,RuBisCO ,Temperature ,Proteins ,GroES ,GroEL ,Pyruvate carboxylase ,enzymes and coenzymes (carbohydrates) ,biological sciences ,Potassium ,biology.protein ,bacteria - Abstract
Both the chaperonin- and MgATP-dependent reconstitution of unfolded ribulosebisphosphate carboxylase (Rubisco) and the uncoupled ATPase activity of chaperonin 60 (groEL) require ionic potassium. The spontaneous, chaperonin-independent reconstitution of Rubisco, observed at 15 but not at 25 degrees C, requires no K+ and is actually inhibited by chaperonin 60, with which the unfolded or partly folded Rubisco forms a stable binary complex. The chaperonin-dependent reconstitution of Rubisco involves the formation of a complex between chaperonin 60 and chaperonin 10 (groES). Formation of this complex almost completely inhibits the uncoupled ATPase activity of chaperonin 60. Furthermore, although the formation of the chaperonin 60-chaperonin 10 complex requires the presence of MgATP, hydrolysis of ATP may not be required, since complex formation occurs in the absence of K+. The interaction of chaperonin 60 with unfolded or partly folded Rubisco does not require MgATP, K+, or chaperonin 10. However, discharge of the complex of chaperonin 60-Rubisco, which leads to the formation of active Rubisco dimers, requires chaperonin 10 and a coupled, K(+)-dependent hydrolysis of ATP. We propose that a role of chaperonin 10 is to couple the K(+)-dependent hydrolysis of ATP to the release of the folded monomers of the target protein from chaperonin 60.
- Published
- 1990
47. A photoaffinity analogue of discodermolide specifically labels a peptide in beta-tubulin
- Author
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Susan Band Horwitz, Paul V. Rucker, Shujun Xia, George A. Orr, Amos B. Smith, and Craig S. Kenesky
- Subjects
G2 Phase ,Models, Molecular ,GTP' ,Paclitaxel ,Stereochemistry ,Peptide ,Antineoplastic Agents ,Photoaffinity Labels ,Biochemistry ,Microtubules ,Microtubule polymerization ,chemistry.chemical_compound ,Lactones ,Microtubule ,Tubulin ,Alkanes ,Humans ,Binding site ,chemistry.chemical_classification ,biology ,Discodermolide ,In vitro ,Protein Structure, Tertiary ,chemistry ,Pyrones ,biology.protein ,Carbamates ,Peptides ,Cell Division ,Protein Binding - Abstract
Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.
- Published
- 2006
48. Direct electrochemistry of porcine purple acid phosphatase (uteroferrin)
- Author
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Gregory J. Wilson, Gerhard Schenk, and Paul V. Bernhardt
- Subjects
Swine ,Iron ,Inorganic chemistry ,Acid Phosphatase ,Biochemistry ,Redox ,Binding, Competitive ,Acid dissociation constant ,Phosphates ,Electron Transport ,chemistry.chemical_compound ,Electrochemistry ,Animals ,Enzyme Inhibitors ,Glycoproteins ,biology ,Arsenate ,Active site ,Purple acid phosphatases ,Hydrogen-Ion Concentration ,Phosphate ,Dissociation constant ,Chemistry ,chemistry ,biology.protein ,Arsenates ,Cyclic voltammetry ,Oxidation-Reduction ,Nuclear chemistry - Abstract
Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (FeIII-FeII f FeIII-FeIII) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Ufo) have been determined. The effect of pH on the redox potentials has been investigated in the range 3 < pH < 6.5, enabling acid dissociation constants for Ufo and its phosphate and arsenate complexes to be calculated.
- Published
- 2004
49. Coordinating Role of His216 in MgATP Binding and Cleavage in Pyruvate Carboxylase
- Author
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Adina-Zada, Abdussalam, primary, Jitrapakdee, Sarawut, additional, Wallace, John C., additional, and Attwood, Paul V., additional
- Published
- 2014
- Full Text
- View/download PDF
50. Roles of Arg427 and Arg472 in the Binding and Allosteric Effects of Acetyl CoA in Pyruvate Carboxylase
- Author
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Adina-Zada, Abdussalam, primary, Sereeruk, Chutima, additional, Jitrapakdee, Sarawut, additional, Zeczycki, Tonya N., additional, St. Maurice, Martin, additional, Cleland, W. Wallace, additional, Wallace, John C., additional, and Attwood, Paul V., additional
- Published
- 2012
- Full Text
- View/download PDF
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