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1
Wawrzynów, Bartosz. "Allosteric regulation of MDM2 protein". Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4507.
Testo completoAbstract (sommario):
The diverse functions of the MDM2 oncoprotein in growth control and tumourigenesis are managed through coordinated regulation of its discrete domains induced by both extrinsic and intrinsic stimuli. A picture of MDM2 is immerging where structurally discrete but interdependent functional domains are linked through changes in conformation. However compelling insights into how this process is carried out have been hindered by inadequate information on the structure and conformation of the full-length protein. The data presented indicates that the C-terminal RING domain of MDM2, primarily responsible of the E3 ubiquitin ligase activity of the protein, has other intriguing functions. The binding of ATP within the RING domain, triggers conformational changes of MDM2 and its main interaction partner – p53. This in effect promotes efficient binding of the p53 tumour suppressor to specific DNA promoter sequences. Moreover, results presented in this thesis demonstrate a novel role for the RING domain of MDM2 in determining the conformation and activity of its N-terminal hydrophobic cleft, the key target of anticancer drugs designed to activate the function of p53 tumour suppressor protein. Specific modulations within the RING domain, affecting Zinc coordination are synonymous with increased binding affinity of the hydrophobic pocket to the transactivation domain of p53 resulting in a gain of MDM2 transrepressor function thus leading to a decrease in p53-dependant gene expression. ThermoFluor measurements and size exclusion chromatography show that changes in the RING motif lack an effect on the overall integrity of the MDM2 protein. The intrinsic fluorescence measurements manifest that these changes generate long range conformational transitions that are transmitted through the core/central acidic domain of MDM2 resulting in allosteric regulation of the N-terminal hydrophobic pocket. Such RING generated conformational changes result in the relaxation of the hydrophobic pocket. Additionally, it is shown that the cooperation between the RING and the hydrophobic cleft in MDM2 has implications in the efficiency of binding of anticancer drugs such as Nutlin by MDM2. Cooperation between the RING and hydrophobic domain of MDM2 to regulate function demonstrates an allosteric relationship and highlights the need to study MDM2 in a native conformation. In essence the presented data demonstrates that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed towards its hydrophobic pocket.
2
Livingstone, Emma Kathrine. "Allosteric Regulation of the First Enzyme in Histidine Biosynthesis". Thesis, University of Canterbury. Chemistry, 2015. http://hdl.handle.net/10092/10470.
Testo completoAbstract (sommario):
The ATP-PRTase enzyme catalyses the first committed step of histidine biosynthesis in archaea, bacteria, fungi and plants.1 As the catalyst of an energetically expensive pathway, ATP-PRTase is subject to a sophisticated, multilevel regulatory system.2 There are two families of this enzyme, the long form (HisGL) and the short form (HisGS) that differ in their molecular architecture. A single HisGL chain comprises three domains. Domains I and II house the active site of HisGL while domain III, a regulatory domain, forms the binding site for histidine as an allosteric inhibitor. The long form ATP-PRTase adopts a homo-hexameric quaternary structure.3,4 HisGS comprises a similar catalytic core to HisGL but is devoid of the regulatory domain and associates with a second protein, HisZ, to form a hetero-octameric assembly.5
This thesis explores the allosteric regulation of the short form ATP-PRTase, as well as the functional and evolutionary relationship between the two families. New insight into the mode allosteric inhibition of the short form ATP-PRTase from Lactococcus lactis is reported in chapter two. A conformational change upon histidine binding was revealed by small angle X-ray scattering, illuminating a potential mechanism for the allosteric inhibition of the enzyme. Additionally, characterisation of histidine binding to HisZ by isothermal titration calorimetry, in the presence and absence of HisGS, provided evidence toward the location of the functional allosteric binding site within the HisZ subunit.
Chapter three details the extensive effort towards the purification of the short form ATP-PRTase from Neisseria menigitidis, the causative agent of bacterial meningitis. This enzyme is of particular interest as a potential target for novel, potent inhibitors to combat this disease. The attempts to purify the long form ATP-PRTase from E. coli, in order to clarify earlier research on the functional multimeric state of the enzyme, are also discussed.
Chapter four reports the investigation of a third ATP-PRTase sequence architecture, in which hisZ and hisGS comprise a single open reading frame, forming a putative fusion enzyme. The engineering of two covalent linkers between HisZ and HisGS from L. lactis and the transfer of the regulatory domain from HisGL to HisGS, is also discussed, in an attempt to delineate the evolutionary pathway of the ATP-PRTase enzymes. Finally, the in vivo activity of each functional and putative ATP-PRTase was assessed by E. coli BW25113∆hisG complementation assays.
3
Cohen, Fiona Rachel. "The allosteric regulation of adenosine receptors". Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309286.
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4
Marshall, Kristin Ann. "Group I aptazymes as genetic regulatory switches". Access restricted to users with UT Austin EID Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3034980.
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5
Cockrell, Gregory Mercer. "New Insights into Catalysis and Regulation of the Allosteric Enzyme Aspartate Transcarbamoylase". Thesis, Boston College, 2013. http://hdl.handle.net/2345/3156.
Testo completoAbstract (sommario):
Thesis advisor: Evan R. KantrowitzThe enzyme aspartate transcarbamoylase (ATCase) is an enzyme in the pyrimidine nucleotide biosynthetic pathway. It was once an attractive target for anti-proliferation drugs but has since become a teaching model due to kinetic properties such as cooperativity and allostery exhibited by the Escherichia coli form of the enzyme. ATCase from E. coli has been extensively studied over that last 60 years and is the textbook example of allosteric enzymes. Through this past research it is understood that ATCase is allosterically inhibited by CTP, the end product of pyrimidine biosynthesis, and allosterically activated by ATP, the end product of the parallel purine biosynthetic pathway. Part of the work discussed in this dissertation involves further understanding the catalytic properties of ATCase by examining an unregulated trimeric form from Bacillus subtilis, a bacterial ATCase that more closely resembles the mammalian form than E. coli ATCase. Through X-ray crystallography and molecular modeling, the complete catalytic cycle of B. subtilis ATCase was visualized, which provided new insights into the manifestation of properties such as cooperativity and allostery in forms of ATCase that are regulated. Most of the work described in the following chapters involves understanding allostery in E. coli ATCase. The work here progressively builds a new model of allostery through new X-ray structures of ATCase*NTP complexes. Throughout these studies it has been determined that the allosteric site is bigger than previously thought and that metal ions play a significant role in the kinetic response of the enzyme to nucleotide effectors. This work proves that what is known about ATCase regulation is inaccurate and that currently accepted, and taught, models of allostery are wrong. This new model of allostery for E. coli ATCase unifies all old and current data for ATCase regulation, and has clarified many previously unexplainable results
Thesis (PhD) — Boston College, 2013
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
6
Brear, Paul. "The search for allosteric inhibitors". Thesis, University of St Andrews, 2013. http://hdl.handle.net/10023/3451.
Testo completoAbstract (sommario):
This thesis describes the development of chemical tools that inhibit the sialidases NanA and NanB from Streptococcus pneumonia. The primary focus was on the discovery of allosteric inhibitors of NanA and NanB, however, promising inhibitors that act by binding at the active site of these enzymes were also investigated. Chapter 1 gives an overview of the use of chemical tools in the field of chemical biology. It focuses in particular on chemical tools that function by the allosteric regulation of their target proteins. The uses, advantages and methods of discovery of allosteric tools are discussed. Finally this chapter introduces the use of serendipitous binders for the discovery of allosteric sites. In particular, the use of CHES to identify novel allosteric sites on the sialidase NanB is proposed. Chapter 2 describes how the ‘hits' from a series of high throughput screens were reanalysed using a wide range of secondary assays to eliminate any false positives that were contaminating the results. This process removed eight of the eleven ‘hits'. Two of the remaining three compounds were then analysed further in an attempt to characterise their binding mode to NanA and/or NanB using modelling and X-ray crystallographic studies. Whilst, it was not possible to confirm the binding mode by X-ray crystallography modelling studies using the modelling software GOLD generated possible binding modes for these inhibitors. A structure activity relationship study was conducted for both compounds in an attempt to generate more potent inhibitors. Chapter 3 moves from the use of high throughput screens to identify hits against NanA and NanB to the use of the serendipitous binding of N-cyclohexyl-2-aminoethanesulfonic acid in the active site of NanB for the development of selective NanB inhibitors. First taurine was identified as the minimum unit of N-cyclohexyl-2-aminoethanesulfonic acid required to bind to the active site of NanB. Taurine was then used as the basis of an optimisation study. This chapter concludes with the identification of 2-(benzylammonio)ethanesulfonate as the next key intermediate in the development of N-cyclohexyl-2-aminoethanesulfonic acid based active site inhibitors of NanB. Chapter 4 follows on from Chapter 3 with the optimisation of 2-(benzylammonio)ethanesulfonate describing the design and synthesis of a wide range of analogues. From these compounds 2-[(3-chlorobenzyl)ammonio]ethanesulfonate was identified as the most potent and selective inhibitor. Detailed analysis of the binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to NanB gave a rationale for its improved inhibitory activity. The increase in inhibition occurred because on binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to the active site of NanB a well coordinated water molecule was displaced. The displacement of this water caused an increase in the flexibility of the enzyme's 352 loop. A detailed study of the flexibility of this loop in response to various N-cyclohexyl-2-aminoethanesulfonic acid based chemical tools was then conducted. The research in chapters 2 and 3 has recently been published. In Chapter 5 a molecule of N-cyclohexyl-2-aminoethanesulfonic acid that binds serendipitously in a previously unmentioned secondary site is elaborated into a ligand, known as Optactin, that binds strongly and selectively at this secondary site. It was then shown that Optactin inhibited NanB by binding at this secondary site. It was therefore concluded that this secondary site was in fact an allosteric site that could be used for the regulation of NanB. Chapter 6 describes the development of a rationalisation for the inhibition of NanB by Optactin. This study included the X-ray crystallographic analysis of the apo-NanB structure and the NanB-Optactin complex under a range of conditions. This was followed by mechanistic studies that identified the point in the catalytic cycle at which Optactin was inhibiting NanB. This chapter concludes with a hypothesis for the mechanism of inhibition of NanB by Optactin.
7
Larsson, Karl-Magnus. "Allosteric regulation and radical transfer in ribonucleotide reductase /". Stockholm : Institutionen för biokemi och biofysik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-251.
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8
Rofougaran, Reza. "DNA precursor biosynthesis-allosteric regulation and medical applications". Doctoral thesis, Umeå : Univ, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1678.
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9
Ali, Mahesheema na. "Allosteric Regulation of Prothrombin Activation by factor Va". Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1462805026.
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10
Schwebach, Christopher L. "Allosteric and Calcium-Dependent Regulation of Human Plastins". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1563329156932864.
Testo completo
11
Paricharttanakul, Nilubol Monique. "Pathway to allostery: differential routes for allosteric communication in phosphofructokinase from Escherichia coli". Texas A&M University, 2004. http://hdl.handle.net/1969.1/1429.
Testo completoAbstract (sommario):
Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated
by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to
the same allosteric site, however, MgADP activates and PEP inhibits the binding of
fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can
differentiate between the two ligands and cause the appropriate response is important for
the understanding of the basis of allosteric regulation.
We studied the interactions between an active site and an allosteric site
(heterotropic interactions) within the protein, and found that each of the four unique
heterotropic interactions is unique and the magnitudes of the coupling free energies for
MgADP activation sum up to 100% that of wildtype EcPFK without homotropic
cooperativity in F6P binding. We took on the kinetic and structural characterization of
phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that
exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et
al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which
are important for the allosteric responses to both MgADP and PEP. Interestingly,
Lys214 is only important in PEP inhibition and not MgADP activation. We can also
differentially disrupt the MgADP heterotropic interactions with the introduction of
G184C within the protein. These results suggest that there are different pathways for
allosteric communication within the enzyme: different paths for MgADP activation and
PEP inhibition, and different paths for each heterotropic interaction with Gly184 being
important for the 33Å MgADP heterotropic interaction.
12
Payne, Marvin A. "Desensitized Phosphofructokinase from Ascaris suum: A Study in Noncooperative Allostery". Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc279174/.
Testo completoAbstract (sommario):
The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.
13
Yu, Peng. "Allosteric regulation of glycerol kinase: fluorescence and kinetics studies". Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/1537.
Testo completoAbstract (sommario):
Glycerol kinase (GK) from Escherichia coli is allosterically controlled by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier protein IIAGlc of the phosphotransferase system. These controls allow glucose to regulate glycerol utilization. Fluorescence spectroscopic and enzyme kinetic methods are applied to investigate these allosteric controls in this study. The linkage between FBP binding and GK tetramer assembly is solved by observation of homo-fluorescence energy transfer of the fluorophore Oregon Green (OG) attached specifically to an engineered surface cysteine in GK. FBP binds to tetramer GK with an affinity 4000-fold higher than to dimeric GK. A region named the coupling locus that plays essential roles in the allosteric signal transmission from the IIAGlc binding site to the active site was identified in GK. The relationship between the coupling locus sequence in Escherichia coli or Haemophilus influenzae GK variants and the local flexibility of the IIAGlc binding site is established by fluorescence anisotropy determinations of the OG attached to the engineered surface cysteine in each variant. The local flexibility of the IIAGlc binding site is influenced by the coupling locus sequence, and in turn affects the binding affinity for IIAGlc. Furthermore, the local dynamics of each residue in the IIAGlc binding site of GK is studied systematically by the fluorescence anisotropy measurements of OG individually attached to each position of the IIAGlc binding site. The fluorescence steady-state anisotropy measurement provides a valid estimation of the local flexibility and correlates well with the crystallographic B-factors. Steady-state kinetics of FBP inhibition shows that the data are best described by a model in which the partial inhibition and FBP binding stoichiometry are taken into account. Kinetic viscosity effects show that the product-release step is not the purely rate-limiting step in the GK-catalyzed reaction. Viscosity effects on FBP inhibition are also discussed.
14
Lu, Jian. "Investigation of allosteric regulation of porcine fructose-1,6-bisphosphatase". [Ames, Iowa : Iowa State University], 2007.
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15
Ivanisevic, Ljubica. "Neutrophin receptors: ligand-binding, activation sites and allosteric regulation". Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18758.
Testo completoAbstract (sommario):
The Trk family of tyrosine kinase receptors and the common p75NTR receptor are neurotrophin receptors. Nerve growth factor (NGF) binds TrkA, brain-derived neurotrophic factor (BDNF) binds TrkB, and neurotrophin-3 (NT-3) binds TrkC. The extracellular domain of the Trk receptor has five subdomains: a leucine-rich motif (D2), two cysteine-rich motifs (D1, D3) and immunoglobulin-like subdomains Ig-C1 (D4) and Ig-C2(D5). The Trk D4 subdomain regulates ligand-independent activation. The TrkA-D5 and TrkB-D5 subdomains regulate cognate ligand binding and Trk activation. However, the p75NTR receptor binds all neurotrophins and regulates ligand affinity and Trk signals. We showed that p75NTR affects Trk ligand - binding and activation of Trks by changing Trk subdomain utilization. When p75NTR is coexpressed, NGF can activate TrkA via the cysteine-1 subdomain (D1), and BDNF can activate TrkB via leucine-rich motif (D2) and cysteine-2 (D3) subdomains. We hypothesized conformational or allosteric regulatory mechanisms. To further study the interactions between ligands and Trks, we examined TrkA binding to NT-3 as a heterologous ligand because these interactions are biologically relevant. We found the TrkA “hot spot” functional docking sites used by NT-3. We demonstrate that TrkA-D5 has partially overlapping but distinct binding and activation “hot spots” for both, NGF and NT-3. Moreover, ligand - binding studies have identified additional NT-3 binding/allosteric site on TrkA-D4. NT-3 binding to both sites induces full agonism. Conversely, the TrkA-D5 NT-3 binding site is partially agonistic, but antagonizes NGF activity. Lasly, we address NT-3 binding and activation sites on the TrkC receptor by raising a monoclonal antibody that recognizes the juxtamembrane-linker domain of the TrkC receptor. This antibody is an artificial TrkC receptor agonist. The epitope of mAb 2B7 defines a previously unknown hot spot of TrkC. Binding to this “hot spot” induces survival but nLa famille de récepteurs de Trk tyrosine kinase et le récepteur p75NTR sont des récepteurs de neurotrophines. Le facteur de croissance nerveuse (NGF) intéragit avec le récepteur TrkA, le facteur neurotrophique dérivé du cerveau (BDNF) intéragit avec le récepteur TrkB et la neurotrophine-3 (NT-3) intéragit avec TrkC. Le domaine extracellulaire du récepteur Trk contient cinq sous-domaines: un motif riche en leucine (D2), deux motifs riches en cysteine (D1, D3) et des sous-domaines de type immunoglobuline Ig-C1(D4) et Ig-C2(D5). Le sous-domaine Trk D4 régule l'activation indépendante de ligand. Les sous-domaines TrkA-D5 et TrkB-D5 régulent la liaison de ligands endogènes ainsi que l'activation du récepteur Trk. Le récepteur p75NTR intéragit avec toutes les neurotrophines et régule l'affinité des ligands et les signaux issues de l'activation du récepteur Trk. Par ailleurs, nous avons démontré que le p75NTR affecte la liaison du ligand au récepteur Trk en changeant l'activation de l'utilisation des sous-domaines. Lorsque le recepteur de p75NTR est coexprimé, le NGF peut activer le récepteur TrkA via le sous-domaine cysteine-1 (D1) et BDNF peut activer TrkB via le motif riche en leucine (D2) ainsi que via le sous-domaine cysteine-2 (D3). Nous avons examiné la liaison d'un ligand hétérologue, NT-3 sur le récepteur TrkA afin d'étudier plus profondément les interactions entre les ligands et le récepteur TrkA. Ces interactions sont biologiquement pertinentes. Pour faire ceci, nous avons tout d'abord identifié les « points chauds » présents sur le récepteur TrkA qui servent des sites d'amarrage fonctionnels du ligand NT-3. Nous avons démontré que le sous domaine TrkA-D5 possède deux points chauds distincts, notamment un point chaud qui sert comme le site d'amarrage et d'activation du NGF et un point chaud qui sert comme le site d'amarrage et d'activation de la NT-3. Toutefois, ces deux sites d'amarrage se chevauchent partiellement. D
16
Huisman, Frances Helen Adam. "Studies into the allosteric regulation of α-isopropylmalate synthase". Thesis, University of Canterbury. Chemistry Department, 2012. http://hdl.handle.net/10092/7599.
Testo completoAbstract (sommario):
α-Isopropylmalate synthase (α-IPMS) catalyses the first committed step in leucine biosynthesis in bacteria, including Neisseria meningitidis and Mycobacterium tuberculosis. It catalyses the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Like many key enzymes in biosynthesis, α-IPMS is inhibited by the end-product of the biosynthetic pathway, in this case leucine. α-IPMS is homodimeric, with monomers consisting of a (β/α)8-barrel catalytic domain, two subdomains and a C-terminal regulatory domain, responsible for binding leucine and providing feedback inhibition for leucine biosynthesis.
The exact mechanism of feedback inhibition in this enzyme is unknown, despite the elucidation of crystal structures with and without leucine bound. This thesis explores the nature of allosteric regulation in α-IPMS, including the effects of the regulatory domain and the importance of structural asymmetry on catalytic activity.
Chapter 2 details the characterisation of wild-type α-IPMS from N. meningitidis (NmeIPMS). This protein was successfully cloned, expressed and purified by metal-affinity and size-exclusion chromatography. NmeIPMS has similar characteristics to previously characterised α-IPMSs, being a dimer and demonstrating substrate binding affinities in the micromolar range. This enzyme has a turnover number of 13s⁻¹ and is sensitive to mixed, non-competitive inhibition by the amino acid leucine. Small angle X-ray scattering experiments reveal that the solution-phase structure of this protein is likely similar to existing crystal structures of other α-IPMSs.
In Chapter 3, substitutions of residues potentially involved in the binding and transmission of the leucine regulatory mechanism are described. Most of these amino acid substituted variants reduce enzyme sensitivity to leucine, and one variant is almost entirely insensitive to this inhibitor. Another of these variants demonstrates an unexpected decrease in substrate affinity, despite the substituted residue being located far from the active site.
The independence of α-IPMS domains is investigated in Chapter 4. The catalytic domains were isolated from NmeIPMS and the α-IPMS from M. tuberculosis (MtuIPMS), and found to be unable to catalyse the condensation of substrates, despite maintaining the wild-type structural fold. Complementation studies with Escherichia coli cells lacking the gene for α-IPMS show that the truncated variants are unable to rescue growth in these cells. Binding of α-KIV in the truncated NmeIPMS variant is much stronger than in the wild-type, and this may be the reason for lack of competent catalysis. A crystal structure was solved for the truncated variant of NmeIPMS and indicates that the regulatory domain is required for proper positioning of large regions of the protein. Two isolated regulatory domains from NmeIPMS were cloned, but with limited success in characterisation.
Finally, Chapter 5 describes substitutions made in MtuIPMS to affect relative domain orientations within the protein. Dimer asymmetry is investigated by substituting residues at the domain interfaces. These substitutions did have some effect on catalysis and inhibition, but did not show any change in average solution-phase structure.
These results are drawn together in the greater context of allostery in general in Chapter 6, along with ideas for future research in this field. This chapter reviews the insights gained into protein structure from this thesis, particularly the importance of residues at protein domain interfaces. The asymmetry in the α-IPMS structure is discussed, along with small-molecule binding regulatory domains.
17
Mendes, Kimberly Rose Marie. "Catalysis and Regulation of the Allosteric Enzyme Aspartate Transcarbamoylase". Thesis, Boston College, 2010. http://hdl.handle.net/2345/2975.
Testo completoAbstract (sommario):
Thesis advisor: Evan R. KantrowitzThe understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of cellular regulation can be related in some manner to protein conformational changes such as the quaternary conformational changes of allosteric enzymes that alter enzyme activity to regulate metabolism. The control of metabolic pathways by allosteric enzymes is analogous to a molecular valve with "on" and "off" positions. In the "off" position, flow through the pathway is severely hindered, while in the "on" position the flow is normal. For a comprehensive understanding of allosteric regulation we must elucidate in molecular detail how the allosteric signal is transmitted to the active site to alter enzyme activity. In this work we use unnatural amino acid mutagenesis to introduce a fluorescent amino acid into the allosteric binding site of aspartate transcarbamoylase (ATCase), the enzyme responsible for regulation of pyrimidine nucleotide biosynthesis. The fluorescence from the amino acid is exquisitely sensitive to the binding of the allosteric effectors ATP, CTP, UTP, and GTP. In particular we show how the asymmetric nature of the allosteric sites of the enzyme are used to achieve regulatory sensitivity over a broad range of mixed heterotropic effector concentrations as is observed in the cell. Furthermore, employing the method of random sampling - high dimensional model representation (RS-HDMR) we derived a model for how ATCase is regulated when all four nucleotides are present at fluctuating concentrations, consistent with physiological conditions. We've discovered the fundamental requirements to induce the allosteric transition to the R state by showing that although ATCase can accept L-asparagine as an unnatural substrate, the transition to the R allosteric state requires the correct positioning of the alpha-carboxylate of its natural substrate L-aspartate. However, linking the functionalities of L-asparagine and carbamoyl phosphate into a single molecule is sufficient to correctly position the bi-substrate analog in the active site to induce the allosteric transition to the R-state. The cooperative nature of ATCase was further investigated through the isolation of a unique quaternary structure of ATCase consisting of two catalytic trimers linked covalently by disulfide bonds. By relieving the quaternary constraints imposed by the bridging regulatory subunits of the native holoenzyme, the flexibility of the c6 subunit significantly enhanced enzyme activity over the native holoenzyme. Unlike the native c3 catalytic subunit, the c6 species displays homotropic cooperativity for L-aspartate demonstrating that, when two catalytic trimers are linked, a binding event at one or more active sites can be transmitted through the molecule to the other active sites in the absence of regulatory subunits. The catalytic reaction of ATCase follows an ordered sequential mechanism that is complicated by the transition from the T state to the R state upon the binding of the second substrate L-aspartate. Acquiring X-ray crystal structures at each step along the pathway has advanced our understanding of the catalytic mechanism, yet R-state structures are difficult to obtain. Using a mutant version of ATCase locked in the R-allosteric state by disulfide bonds we captured crystallographic images of ATCase in the R state bound to the true substrates (CP and Asp), products (CA and Pi), and in the process of releasing the final product (Pi) prior to reversion of the molecule to the T state. These structures depict the steps in the catalytic cycle immediately before the catalytic reaction occurs, immediately after the reaction, and after the first product has been released from the active site. This work also focuses on developing allosteric inhibitors of the enzyme fructose-1,6-bisphosphatase (FBPase), one of the enzymes responsible for regulation of the gluconeogenesis pathway. Inhibitors of FBPase could serve as potential therapeutic agents against type-2 diabetes
Thesis (PhD) — Boston College, 2010
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
18
Zheng, Yunan. "Study of Allosteric Regulation of Escherichia coli Aspartate Transcarbamoylase". Thesis, Boston College, 2013. http://hdl.handle.net/2345/3683.
Testo completoAbstract (sommario):
Thesis advisor: Evan R. KantrowitzFor nearly 60 years the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and 5 X-ray structures determined in the absence and presence of a Mg2+ concentration within the physiological range. In the presence of 2 mM divalent cations (Mg2+, Ca2+, Zn2+) CTP does not significantly inhibit the enzyme while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor in vivo of ATCase is the combination of CTP, UTP and a M2+ cation and the actual allosteric activator is ATP and M2+ or ATP, GTP and M2+. The structural data reveals that two NTPs can bind to each allosteric site with a Mg2+ ion acting as a bridge between the triphosphates. Thus the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal the catalytic chains undergo essentially no alternations, however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal regions of the regulatory chains exist in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed
Thesis (MS) — Boston College, 2013
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
19
Hines, Justin Keith. "Allosteric regulation of mammalian and bacterial fructose-1,6-bisphosphatases". [Ames, Iowa : Iowa State University], 2007.
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20
Cheng, Cecilia Yuen-Man. "Dissecting the allosteric regulation of PKA-I alpha activation". Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3355645.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed June 23, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 200-214).
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Purohit, Rahul. "The Mechanism of Allosteric Regulation in Soluble Guanylate Cyclase". Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/333219.
Testo completoAbstract (sommario):
Nitric oxide (NO), a reactive diatomic gas and a potent signaling molecule, is required for proper cardiovascular functioning. Soluble guanylate cyclase (sGC), a heterodimeric heme protein, is the key intracellular NO receptor protein which, upon NO binding, undergoes conformational changes leading to catalysis and the cGMP signaling cascade. Several small molecules that allosterically stimulate sGC have been developed for treatment of pulmonary hypertension, but little is known about their binding site or how they stimulate activity. This dissertation describes experiments designed to uncover the molecular basis for signal transduction in sGC by NO and small molecule stimulators. The crystal structure of the α-subunit PAS domain from Manduca sexta (Ms) sGC was solved at 1.8 Å resolution revealing the expected PAS fold but with an additional β strand and a shorter Fα helix. CO binding measurements on different Ms sGC N-terminal constructs and the β₁ (1-380) construct revealed that the α-subunit keeps the β₁ H-NOX domain in an inhibited conformation and this inhibition is relieved by removal of the α-subunit or by addition of stimulatory compounds such as compound YC-1. Linked-equilibria measurements on the N-terminal constructs show that YC-1 binding affinity is increased in the presence of CO. Surface plasmon resonance (SPR) studies on the in-vitro biotinylated constructs showed that YC-1 binds near or directly to the β₁ H-NOX domain. Computational and mutational analysis of the β₁ H-NOX domain revealed a pocket important in allostery and drug action. Finally, we show that the coiled coil domain plays an important role in allosteric regulation of the β₁ H-NOX domain and possibly in signal transduction. Our data are consistent with a model of allosteric activation in which the α-subunit and the coiled coil domains function to keep heme in a low affinity conformation while YC-1 binding to the β₁ H-NOX domain switches heme to a high affinity conformation, and sGC to its high activity form.
22
Mittelstädt, Gerd Horst. "Allosteric regulation of the adenosine triphosphate phosphoribosyltransferase from campylobacter jejuni". Thesis, University of Canterbury. Chemistry, 2015. http://hdl.handle.net/10092/10799.
Testo completoAbstract (sommario):
The enzyme adenosine triphosphate phosphoribosyltransferase (ATP-PRT)
catalyses the first reaction of the histidine biosynthetic pathway. ATP-PRT
also represents a metabolic control point, directing the flux of metabolites
through this energetically expensive pathway. Two distinctly different forms
of ATP-PRT exist, the long form and the short form, which differ in the
presence of a C-terminal regulatory domain. In the short form, where this
domain is absent, it is functionally replaced by a regulatory protein, called
HisZ. ATP-PRT activity is modulated by two layers of regulation: active site
inhibition by adenosine monophosphate, which reflects cellular energy levels, and pathway end product feedback inhibition by histidine. In the long form ATP-PRT histidine binds to the allosteric site at the regulatory domain, but the exact nature of the inhibitory mechanism is still debated.
This thesis characterises a new member of the ATP-PRT long form from Campylobacter jejuni (CjeATP-PRT) and investigates the molecular mechanisms involved in the feed back inhibition by histidine.
Chapter 2 describes the characterisation of the CjeATP-PRT including
a detailed description of its crystal structure. The C. jejuni enzyme is similar
to the previously described enzymes of the ATP-PRT long form, but exists
only as hexameric species under experimental conditions, which contradicts
previous assumptions that the hexamer is exclusively inactive.
Chapter 3 investigates the catalytic apparatus of CjeATP-PRT by separating
the catalytic and regulatory domains of the enzyme for individual study. The isolated catalytic portion of the enzyme, the CjeATP-PRT Core mutant, forms a dimeric species with very limited catalytic capabilities but high substrate and product affnities. The CjeATP-PRT Core characteristics suggest that it exists in a permanently inhibited conformation, highlighting
the requirement of the regulatory domain not only for feedback regulation
but also for enzyme function. Additionally this supports the evolutionary
need for the recruitment of a regulatory apparatus.
In chapter 4 a potential intramolecular communication pathway from
the allosteric to the active site is probed by the generation of several single site mutations. One of these, CjeATP-PRT R216A, is completely insensitive to histidine inhibition, although this ligand is still able to bind at the allosteric
site, which is consistent with the involvement of R216 in the allosteric signal
communication. The catalytic abilities of CjeATP-PRT R216A are largely
impaired, leading to the assumption that this mutation causes a permanent
inhibitory response.
In summary this thesis supports the existence of a simple physical regulatorymechanism for the feedback inhibition of the ATP-PRT long form,
the change between two different hexamer conformations depending on the
presence of the allosteric effector.
23
Abdelrahman, Mostafa Hamed. "Design, synthesis and SAR of novel allosteric modulators of the Cannabinoid CBI receptor". Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=159203.
Testo completoAbstract (sommario):
We report on the design, synthesis, and structure activity relationship studies of novel Org 27569 analogues as potential allosteric modulators of the CB1 receptors. We also investigated by computer modelling the possible location of the allosteric site on CB1 and the binding confirmation of the allosteric ligands. Docking of the synthesised molecules is also performed and the results are compared to the results of the biological bioassays. The synthesis of non-fused indole analogues of Org 27569 is described. These analogues were systematically varied to study the importance of key functional groups for CB1 allosteric activity. It was found that the two NH groups of the indole derivatives are required for activity. Activity is also significantly improved for analogues possessing a hydroxymethyl group or a hydrophobic chain at position 3 of the indole moiety. SAR analysis also shows that the presence of a dialkylamino group at the para-position on the aromatic side chain further improves the activity. Conformationally restricted analogues (fused indoles) of Org 27569 were prepared to determine the possible binding conformation of Org 27569. An analogue having the two NH groups directed in the same direction exhibited a moderate ability to enhance CP55,940 affinity and gave significant decrease in [35S]GTPγS binding at 1μM, indicating the possible binding conformation for the Organon derivatives. Molecular modelling studies allowed locating a possible binding pocket for the CB1 allosteric ligands. The study described here should help the design of ligands of the CB1 allosteric site that possess higher biological activities and specificities. The results should pave the way for the discovery of the anti-obesity drugs of the future.
24
Cross, Penelope Jane. "Unravelling the Evolution of Allosteric Regulation in 3-Deoxy-D-arabino-heptulosonate 7-phosphate Synthase". Thesis, University of Canterbury. Chemistry, 2012. http://hdl.handle.net/10092/6823.
Testo completoAbstract (sommario):
The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first reaction in the shikimate pathway, leading to the biosynthesis of aromatic compounds including the aromatic amino acids. The catalytic activity of DAH7PS is regulated through feedback inhibition and is the major control point for the pathway. DAH7PSs are divided into two families, type I and type II, based on molecular weight and amino acid sequence. Type I DAH7PSs can be further divided based on sequence similarity. All DAH7PS enzymes with their crystal structures solved share a basic (β/α)₈-barrel fold in which the key catalytic components are housed. Furthermore, all structurally characterised DAH7PSs, except Pyrococcus furiosus DAH7PS (PfuDAH7PS) and Aeropyrum pernix DAH7PS, have recruited extra structural motifs that are implicated in allosteric regulation. However, there are significant differences in the additional structural elements.
This thesis investigates the hypothesis that the diverse regulation strategies for controlling DAH7PS activity have evolved by domain recruitment, whereby regulatory domains have been added to the catalytic barrel.
Chapter 2 describes the functional characterisation of the type Iβ Thermotoga maritima DAH7PS (TmaDAH7PS), and the exploration of its response to inhibitors. The catalytic activity of TmaDAH7PS was found to be substantially inhibited by tyrosine (Tyr) and to a lesser extent, phenylalanine (Phe). The putative regulatory domain previously identified as a ferredoxin-like domain was recognised as an aspartate kinase-chorismate-mutase-tyrA (prephenate dehydrogenase) or ACT domain.
Chapter 3 describes the characterisation of TmaDAH7PS with the N-terminal domain removed. The truncated enzyme was found to be more catalytically active than wild-type TmaDAH7PS and insensitive to inhibition by the aromatic amino acids, Tyr, Phe and tryptophan. Apart from the truncation of the ACT domain, the crystal structure of truncTmaDAH7PS showed no major changes to the monomer structure when compared to wild-type TmaDAH7PS. However, truncTmaDAH7PS crystallises as a dimer, unlike wild-type TmaDAH7PS.
In Chapter 4, the solution of the crystal structure of TmaDAH7PS with Tyr bound is presented. Tyr binding was shown to induce a significant conformational change, and Tyr is observed to bind at the interface between the ACT domains from two diagonally located monomers of the tetramer. The major reorganisation of the regulatory domain with respect to the barrel observed in the crystal structure, was confirmed by small angle X-ray scattering. The closed conformation adopted by the protein on Tyr binding physically gates the neighbouring barrel and blocks substrate entry into the active site.
Chapter 5 explores the interactions between TmaDAH7PS and the allosteric inhibitor, Tyr. The residues His29 and Ser31, which form hydrogen bonds with the hydroxyl moiety of the Tyr ligand, were examined for their impact on the sensitivity and selectivity of the enzyme for the inhibitors Tyr and Phe. The hydroxyl side chain of Ser31 was found to be important for both the preferential inhibition by Tyr over Phe and the inhibitory mechanism. His29 (the hydrogen-bonding partner of Ser31) appears to play a secondary role in determining ligand selectivity and the relative positioning of these two residues is crucial to the inhibition of the enzyme.
Chapter 6 evaluates the transferability of allosteric control of catalytic activity. The ACT domain of TmaDAH7PS was fused onto the barrel of the unregulated PfuDAH7PS. This chimeric enzyme was found to be catalytically active, inhibited by Tyr (although less sensitive) and preliminary crystallographic results show inhibition occurs via the same conformational change observed for wild-type TmaDAH7PS.
25
Kamadurai, Hari Bascar. "Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1172778957.
Testo completo
26
Gonzalez, Walter G. "Protein-Ligand Interactions and Allosteric Regulation of Activity in DREAM Protein". FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/2503.
Testo completoAbstract (sommario):
Downstream regulatory antagonist modulator (DREAM) is a calcium sensing protein that co-assembles with KV4 potassium channels to regulate ion currents as well as with DNA in the nucleus, where it regulates gene expression. The interaction of DREAM with A-type KV4 channels and DNA has been shown to regulate neuronal signaling, pain sensing, and memory retention. The role of DREAM in modulation of pain, onset of Alzheimer’s disease, and cardiac pacemaking has set this protein as a novel therapeutic target. Moreover, previous results have shown a Ca2+ dependent interaction between DREAM and KV4/DNA involving surface contacts at the N-terminus of DREAM. However, the mechanisms by which Ca2+ binding at the C-terminus of DREAM induces structural changes at the C- and N-terminus remain unknown. Here, we present the use of biophysics and biochemistry techniques in order to map the interactions of DREAM and numerous small synthetic ligands as well as KV channels. We further demonstrate that a highly conserved network of aromatic residues spanning the C- and N-terminus domains control protein dynamics and the pathways of signal transduction on DREAM. Using molecular dynamics simulations, site directed mutagenesis, and fluorescence spectroscopy we provide strong evidence in support of a highly dynamic mechanism of signal transduction and regulation. A set of aromatic amino acids including Trp169, Phe171, Tyr174, Phe218, Phe235, Phe219, and Phe252 are identified to form a dynamic network involved in propagation of Ca2+ induced structural changes. These amino acids form a hydrophobic network connecting the N- and C-terminus domains of DREAM and are well conserved in other neuronal calcium sensors. In addition, we show evidence in support of a mechanism in which Ca2+ signals are propagated towards the N-terminus and ultimately lead to the rearrangement of the inactive EF-hand 1. The observed structural motions provide a novel mechanism involved in control of the calcium dependent KV4 and DNA binding. Altogether, we provide the first mechanism of intramolecular and intermolecular signal transduction in a Ca2+ binding protein of the neuronal calcium sensor family.
27
Nilsson, Per. "Allosteric Regulation of mRNA Metabolism : -Mechanisms of Cap-Dependent Regulation of Poly(A)-specific Ribonuclease (PARN)". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8647.
Testo completo
28
Davies, Andrew. "Investigating the selectivity and mechanism of allosteric regulation in α-IPMS enzymes". Thesis, University of Canterbury. Department of Chemistry, 2015. http://hdl.handle.net/10092/10849.
Testo completoAbstract (sommario):
Enzymes are nature’s wizards: balanced delicately on the margin of order and entropy, they perform chemical reactions and syntheses at rates and yields human chemists can only dream of. Many possess exquisite control mechanisms to keep the flow of metabolites through our cells precisely regulated. This work explores the regulation mechanism of α-isopropylmalate synthase (α-IPMS).
The branched-chain amino acid biosynthetic pathways in bacteria are of interest as novel antibiotic targets. α-IPMS catalyses the first committed step in the pathway to form leucine, an essential amino acid. It performs the Claisen condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Almost all previously characterised α-IPMS enzymes are feedback regulated by leucine, the end-product of this pathway.
This study uses the α-IPMS enzymes from two pathogenic species, Myco- bacterium tuberculosis and Neisseria meningitidis (MtuIPMS and NmeIPMS, respectively). These enzymes are homodimeric in solution, and have a catalytic dimer of (β/α)8 barrels. This is connected via two more subdomains to a dimerised C-terminal regulatory domain, where leucine binds. The crystal structures of MtuIPMS with and without leucine bound are almost identical. Thus, we do not yet fully understand the mechanisms by which leucine is
recognised, nor how the allosteric signal is conducted ̴ 50 Å from the regulatory domain to the active site, and how this disrupts catalysis.
Chapter 2 explores the residues responsible for recognising and binding leucine. We use insights from the partial crystal structure of a similar enzyme in Leptospira interrogans, citramalate synthase (CMS). CMS catalyses a similar reaction to α-IPMS: the condensation of AcCoA and α-ketobutyrate (α-KB) to form citramalate, as the first step in isoleucine production in this organism. CMS is feedback regulated by isoleucine just as α-IPMS is regulated by leucine. CMS also shares a very similar overall structure to α-IPMS, and four conserved residues in each enzyme were identified as being responsible for binding the allosteric effector. In previous work, Tyler Clarke1 mutated each of the four MtuIPMS residues to the corresponding residue from LiCMS in an attempt to make an isoleucine-regulated MtuIPMS. While one mutant did show an increased sensitivity to the related amino acid norvaline, none of these mutations by themselves were sufficient to create an isoleucine-sensitive MtuIPMS. This work found that by using certain combinations of these mutations, we were able to create isoleucine-inhibited α-IPMS enzymes.
Dr. Wanting Jiao has been using molecular dynamics simulations to identify the residues important for allosteric signal propagation and disrupting catalysis in NmeIPMS . Chapter 3 details several of these residues which we have mutated, and presents the preliminary results of activity and inhibition studies on the mutant enzymes.
Chapter 4 summarises our findings and outlines the work required to further our understanding of the allosteric control systems studied here. Adapting the power of enzymes to contribute to the development of green chemistry, biosensors, and new antibiotics may prove to be one of the greatest opportunities ahead of modern chemistry.
29
Sintra, Pisco João. "Studies on the mechanism of allosteric regulation of M. tuberculosis ATP-phosphoribosyltransferase". Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10046004/.
Testo completoAbstract (sommario):
Allosteric regulation is an efficient way of controlling enzymatic activity. In Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, ATPphosphoribosyltransferase (ATP-PRT) catalyses the first and committed step of the biosynthesis of L-histidine (L-His). L-His biosynthetic pathway is essential for Mtb and is absent in humans, making ATP-PRT an attractive target for the development of novel antibiotics. ATP-PRT is a hexamer in solution and, like other enzymes regulated via ferrodoxin-like (FL) domains, interconverts between an open active and a closed inactive conformation. Binding of L-His, its feedback allosteric inhibitor, shifts the equilibrium towards the closed state. The exact mechanisms of allosteric control of ATP-PRT activity are currently poorly understood. Likewise, it is unknown if perturbations in the allosteric regulation of ATP-PRT would be detrimental to Mtb. In this work, I characterised the allosteric regulation of ATP-PRT by L-His employing enzymology, biophysics, native mass spectrometry and X-ray crystallography. Using analogues of L-His, I showed that the allosteric inhibition is specific for L-His. A new targeted compound screen for possible allosteric regulators was developed and led to the discovery of 3-(2-Thienyl)-L-alanine (TIH) as an allosteric activator. Kinetic and structural analyses revealed uncoupling between ATP-PRT activity and conformational changes. In order to understand the molecular basis of allosteric regulation of ATP-PRT by L-His, I generated and analysed ATP-PRT variants with point mutations in the allosteric site. These mutations had a direct effect on ATP-PRT stability, activity and allosteric regulation by L-His and TIH. Overall, the results of this work show a complex allosteric regulation of ATPPRT by L-His while revealing the possibility of allosteric activation. Results with the ATP-PRT variants will guide us towards a detailed characterization of the allosteric control of ATP-PRT and its importance for Mtb, which might have implications to other FL domain-containing enzymes and prove useful for drug discovery.
30
Hu, Gang. "Investigation of the importance and structural basis of allosteric regulation of yeast NAD⁺-specific isocitrate dehydrogenase : a dissertation /". San Antonio : UTHSC, 2006. http://proquest.umi.com/pqdweb?did=1251894411&sid=1&Fmt=2&clientId=70986&RQT=309&VName=PQD.
Testo completo
31
Martinez, Gilbert. "Allosteric regulation of clc transport proteins by cytoplasmic domains and conserved CBS Domains /". May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
Testo completo
32
Yeo, Reichelle. "Redox Regulation of Protein Kinase B/Akt Function by an Allosteric Disulphide Bond". Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/20816.
Testo completoAbstract (sommario):
Most proteins in nature are chemically modified after they are made to control how, when and where they function. One type of chemical modification is the cleavage of disulphide bonds that link pairs of cysteine residues in the polypeptide chain. These cleavable bonds are known as allosteric disulphides. From an analysis of labile disulphide bonds in all protein structures from the Protein Data Bank (PDB), my colleagues and I identified a potential allosteric disulphide in the serine/threonine protein kinase B/Akt; linking cysteine residues 60 and 77 in the N-terminus pleckstrin homology (PH) domain. Akt plays a central role in glucose metabolism, cell survival and angiogenesis and is often hyper-activated in cancer cells. Akt is activated at the plasma membrane via binding to phosphatidylinositol-3,4,5-trisphosphate (PIP3) through its PH domain. Dissociation of Akt from the plasma membrane leads to PH domain-mediated autoinhibition of the kinase by a mechanism that is currently unknown. I hypothesised that the PH domain Cys60–Cys77 disulphide is an allosteric bond that regulates autoinhibition and inactivation of the kinase. To elucidate the role of the Cys60–Cys77 disulphide bond in Akt function, wild-type and reduced (Cys60 and/or Cys77 substituted with Ser) PH domain or full-length Akt mutants were analysed for PIP3 plasma membrane binding, Akt phosphorylation and Akt downstream substrate activation, transformation of fibroblasts, and angiogenesis, survival and development of zebrafish. Ablation of the Cys60–Cys77 disulphide bond did not appreciably affect binding of recombinant PH domain to PIP3, but markedly impaired insulin-stimulated binding of full-length Akt to the plasma membrane of adipocytes. Ablation of the Cys60–Cys77 disulphide bond had mixed effects on insulin-stimulated phosphorylation of Akt in fibroblasts. The Cys60Ser mutant was phosphorylated to the same extent as the wild-type, while the Cys77Ser mutant was poorly phosphorylated. Wild-type but not disulphide mutant Akt induced transformation of fibroblasts, indicating an oncogenic role for oxidised but not reduced Akt. Expression of disulphide mutant Akt in zebrafish increased the induction of angiogenesis and development of embryos but did not affect zebrafish survival. My findings imply that the Cys60–Cys77 disulphide bond in the PH domain of Akt is an allosteric disulphide involved in autoinhibition and functioning of the kinase.
33
Zimanyi, Christina Marie. "Structural studies of allosteric regulation in the class Ia Ribonucleotide reductase from Escherichia coli". Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/82330.
Testo completoAbstract (sommario):
Thesis (Ph. D. in Biological Chemistry)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication and repair. The E. coli class Ia enzyme requires two subunits to catalyze the radical-based reduction reaction. [beta]2 houses a diferric-tyrosyl radical cofactor and [alpha]2 contains the active site and two allosteric effector binding sites. Allosteric control of RNR fine-tunes both the relative ratios (via substrate specificity regulation) and the total amount (via activity regulation) of deoxyribonucleotides (dNTPs) in the cell. The molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the [alpha]2 and [beta]2 subunits interact. Here, we present the structure of a complex between the [alpha]2 and [beta]2 subunits in the presence of negative activity effector dATP, revealing an [alpha]4[beta]4 ring-like structure. Using electron microscopy (EM), small-angle X-ray scattering (SAXS), and analytical ultracentrifugation (AUC) we show how activity regulation is achieved by modulating the distributions of active [alpha]2[beta]2 and inhibited [alpha]4[beta]4, an interconversion that requires dramatic subunit rearrangements. The X-ray crystal structure of the dATP-inhibited RNR and a second structure obtained using a mechanism based inhibitor reveal that [alpha]4[beta]4 rings can interlock to form an ([alpha]4[beta]4)2 megacomplex. We use SAXS to understand the solution conditions that contribute to the observed concatenation and present a mechanism for the formation of these unusual structures. We also present the first X-ray crystal structures of [alpha]2 with ATP or dATP bound at both allosteric sites, and discuss how observed differences in their binding influence the modulation between [alpha]2[beta]2 and [alpha]4[beta]4. Finally, we present structures that comprise a full set of cognate substrate/specificity effector pairs bound to the E. coli class Ia RNR. These structures allow us to describe how binding of dNTP effectors at the specificity site promotes binding of a preferred substrate. With these structural data, we describe in molecular detail, how the binding of allosteric effectors influences RNR activity and substrate specificity.
by Christina Marie Zimanyi.
Ph.D.in Biological Chemistry
34
Jurica, Melissa Sue. "I. Structures of intron encoded homing endonucleases ; and, II. Allosteric regulation of pyruvate kinase /". Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/5002.
Testo completo
35
Helmstaedt, Kerstin. "Yeast Chorismate Mutase: Molecular Evolution of an Allosteric Enzyme". Doctoral thesis, [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=967078385.
Testo completo
36
Goupil, Eugénie. "Biased allosteric regulation of the Prostaglandin F2α receptor: from small molecules to large receptor complexes". Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=117082.
Testo completoAbstract (sommario):
G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors, and thus some of the most important targets for drug discovery. By binding to the orthosteric site where endogenous ligands bind, agonists and antagonists differentially modulate signals sent downstream from these receptors. New evidence suggests that GPCRs possess topographically distinct or allosteric binding sites, which may differentially modulate agonist- and antagonist-mediated responses to selectively affect distinct signalling pathways coupled to the same receptor. These sites may either positively or negatively regulate receptor activity, depending on the pathway in question, and thus can act as biased ligands, leading to functional selectivity (or ligand-directed signalling). Another way of allosterically regulating GPCR signalling is through receptor oligomerization, which has recently emerged as a common mechanism for regulating receptor function. The GPCR for prostaglandin F2α FP, is implicated in many important physiological responses, such as parturition, smooth muscle cell contraction and blood pressure regulation. Therefore, evaluating the potential use of allosteric modulators of FP to fine-tune PGF2α-mediated signals, as well as generating a better understanding of its putative oligomerization partners would be of significant pharmacological and clinical interest. In this thesis, I studied the impact of modulating, in both heterologous (HEK 293 cells) and homologous (osteoblast, myometrial or vascular smooth muscle cells) systems, downstream cellular responses of FP by 1) an orthosteric, but biased ligand, previously characterized as a neutral antagonist 2) an allosteric molecule, designed based on the extracellular domains of FP, which had biased signalling properties and, 3) heterodimerization with a receptor partner, the angiotensin II type I receptor, where I demonstrated the asymmetrical organization of this new signalling unit both in vitro and in vivo. Overall, my thesis unveils important roles for biased, allosteric ligands and receptor oligomerization in modulating FP signalling. This work also demonstrates the importance of understanding distinct receptor conformations, and their effects on cellular responses, which are adopted when GPCRs are allosterically modulated, to design better therapeutics with improved efficacy profiles and reduced side effects.Les récepteurs couplés aux protéines G (RCPGs) repésentent la plus grande famille des récepteurs exprimés à la membrane plasmique et sont aussi considérés comme étant des cibles imporantes dans la découverte de nouveaux médicaments. Lorsque des agonistes ou antagonistes se lient au site de liaison endogène d'un RCPG, ou site orthostérique, ces derniers peuvent en moduler les signaux déployés en aval. De nouvelles évidences suggèrent que les RCPGs possèdent des sites de liaison topographiquement distincts des sites orthostériques, appelés sites allostériques. Ces sites allostériques sont suspectés de sélectivement réguler les différents sentiers de signalisation induits lorsque les récepteurs sont liés, de manière concomitante, par des agonistes ou antagonites. De plus, ces sites allostériques peuvent réguler de manière positive ou négative les différentes activités d'un RCPG, et donc être considérés comme étant des ligands biasés, menant à ce qui est appelé la sélectivité fonctionnelle (aussi connue sous le nom de signalisation dirigée par le ligand). Une autre façon de réguler les signaux des RCPGs, qui est présentement vue comme un autre mécanisme contrôlant leur fonction, est l'oligomérization de ces derniers avec d'autres RCPGs, phénomène pouvant être aussi considéré comme de l'allostérisme. Le RCPG pour la prostaglandine F2α, FP, est impliqué dans plusieur réponses physiologiques d'importance, telles la parturition, la contraction des cellules musculaires lisses, ou même la régulation de la pression sanguine. En somme, l'évaluation des différentes façons par lesquelles les signaux de FP peuvent être altérés, soit par l'utilisation d'un modulateur allostérique, soit par l'oligomérisation avec d'autres RCPG, est considérée primordiale d'un point de vue clinique et pharmacologique.Dans cette thèse, j'ai étudié les impacts de la modulation des réponses en aval de FP dans des systèmes hétérologues (cellules HEK 293) ou homologues (cellules ostéoblastiques, myométriales ou musculaires lisses vasculaires), lorsque celui-ci était régulé par 1) un ligand orthostérique, mais à fonctions biaisées, connu précédemment comme un antagoniste neutre, 2) une molecule allostérique, inspirée des domaines extracellulaires de FP, étant aussi capable de propriétés de signalisation biaisées et par 3) l'hétérodimérisation de FP avec un autre récepteur- « partenaire », le récepteur à l'angiotensine II, pour lequel j'ai démonté la présence d'une organisation asymétrique de cette nouvelle « unité » de signalisation, in vitro et in vivo.De manière générale, ma thèse soulève le rôle des ligands biaisés ou allostériques, ainsi que de l'oligomérisation, dans la modulation des signaux cellulaies dirigés par FP. Le travail accompli démontre aussi l'importance de comprendre les différentes conformations, et leurs effets sur les réponses cellulaires, prises quand les RCPGs sont modulés, afin de générer de meilleurs médicaments ayant une meilleure efficacité, mais aussi des effets secondaires plus minimes.
37
De, Zutter Julie Kelley. "Allosteric Regulation of Recombination Enzymes E. coli RecA and Human Rad51: A Dissertation". eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/192.
Testo completoAbstract (sommario):
ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA.
Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament.
The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes.
In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.
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Lechtenberg, Bernhard Clemens. "Thrombin allostery and interactions probed by NMR spectroscopy and crystallography". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610702.
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Jonna, Venkateswara Rao. "Class I Ribonucleotide Reductases : overall activity regulation, oligomerization, and drug targeting". Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-133817.
Testo completoAbstract (sommario):
Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity. The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR. Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively. The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group. We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.
40
Liu, Xuying. "Structure and Regulation of Aspartate Pathway Enzymes and Deuteration Effects on Protein Structure". University of Toledo / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1207946924.
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Leung, Daisy W. "Biochemical and biophysical characterization of the allosteric equilibrium of the Wiskott-Aldrich Syndrome protein". Access to abstract only; dissertation is embargoed until after 12/20/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=131.
Testo completo
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Ke, Wei. "Mechanisms of β- lactamase Inhibition and Heterotropic Allosteric Regulation of an Engineered β- lactamase-MBP Fusion Protein". Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1301692251.
Testo completo
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Pennella, Mario Antonio. "Metal specificity and the mechanism of allosteric regulation in metal-sensing metal-responsive transcriptional repressors Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR". Texas A&M University, 2003. http://hdl.handle.net/1969.1/2303.
Testo completoAbstract (sommario):
The metal-responsive transcriptional repressors of the SmtB/ArsR family repress
the expression of their respective operons in the absence of metal and are released from
the operator/promoter region when metal ions bind, thus allowing RNA polymerase to
bind and transcribe the operon, which encodes genes involved in homeostasis and
resistance. To elucidate the determinants of metal ion selectivity, comparative metalbinding
and DNA-binding properties of S. aureus CzrA and M. tuberculosis NmtR were
characterized. The structure of the metal coordination complexes of CzrA and NmtR
reveal that CzrA forms a 4-coordinate, tetrahedral complex with both Zn(II) and Co(II)
potent regulators of czr operator/promoter (O/P) binding in vitro and de-repression in
vivo. In contrast, NmtR adopts 5- or 6-coordinate complexes with Ni(II) and Co(II), the
strongest allosteric regulators of nmt O/P binding in vitro and de-repression in vivo.
Zn(II), a non-inducer in vivo and poor regulator in vitro, binds NmtR with high affinity
and forms a non-native 4-coordinate complex. These studies suggest that metal
coordination geometries (number), not metal binding affinities, are primary determinants
of functionality.
To gain molecular insight into the mechanism of allosteric regulation of O/P
binding by metal ions, NMR and X-ray crystallographic studies of apo- and zinc forms
of CzrA, and another ArsR/SmtB zinc sensor, Synechococcus PCC7942 SmtB, were
performed. These studies showed that formation of the metal chelate drives a quaternary
structural switch mediated by an intersubunit hydrogen-binding network that originates
with the nonliganding Nε2 face of His97 in CzrA (His117 in SmtB) that stabilizes a low
affinity DNA-binding conformation.
Mutagenesis experiments reveal that substitution of D84 and H97 in CzrA,
results in the formation of higher coordination number complexes that are nonfunctional
in driving zinc-mediated allosteric regulation of DNA binding. In contrast, conservative
mutations of H86 and H100 in CzrA bind Co(II) or Zn(II) in a tetrahedral manner, albeit
with greatly reduced affinity, and allosterically regulate O/P binding with significant
lower coupling free energies compared to wild-type CzrA. These findings further
reinforce the notion that metal coordination geometry is the primary determinant for
functional sites in metal-sensing transcriptional repressors.
44
Quinlan, Robert Jason. "An investigation into the role of protein-ligand interactions on obligate and transient protein-protein interactions". Texas A&M University, 2004. http://hdl.handle.net/1969.1/1430.
Testo completoAbstract (sommario):
Protein-ligand and protein-protein interactions are critical to cellular function. Most cellular metabolic and signal tranduction pathways are influenced by these interactions, consequently molecular level understanding of these associations is an important area of biochemical research. We have examined the thermodynamics of several protein-protein associations and the protein-ligand interactions that mediate them.
Using Fluorescence Correlation Spectroscopy, we have examined the putative interaction between pig heart malate dehydrogenase (MDH) and citrate synthase (CTS). We demonstrate a specific, low-affinity interaction between these enzymes. The association is highly polyethylene glycol (PEG)-dependent, and at high concentrations of NaCl or PEG, non-specific aggregates are formed. We demonstrate that oxaloacetate, the intermediate common to both CTS and MDH, induces the association at concentrations below the Km of CTS, suggesting that the open conformation of CTS is involved in the association.
Using several biophysical techniques, we have examined the subunit associations of B. stearothermophilus phosphofructokinase (PFK). We demonstrate that the inhibitor bound conformation of the enzyme has reduced subunit affinity. The kinetics and thermodynamics of the phosphoenolpyrvuate (PEP)-induced dissociation of PFK have been quantified. Binding substrate, fructose-6-phosphate (F6P), stabilizes the enzyme to inhibitor-induced dissociation by 132-fold. These data suggest that subunit associations may play a role in the allosteric inhibition of PFK by PEP.
The thermodynamics of the protein-ligand associations and allosteric inhibition of E. coli phosphofructokinase have been examined using intrinsic fluorescence and hydrostatic pressure. Both ligand-binding affinity and PEP inhibition are diminished by pressure, whereas substrate-binding affinity for inhibitor-bound enzyme is pressure-insensitive. Larger entropic than enthalpic changes with pressure lead to the overall reduction in free energies.
Using a fluorescence-based assay, we have developed a series of baroresistant buffer mixtures. By combining a buffer with acid dissociation of negative volume with a buffer of positive volume, a pressure-resistant mixture is produced. Alteration of the molar ratio of the two component buffers yields mixtures that are pressure-insensitive at pH values around neutrality.
45
Fish, Eric W. "GABAA positive modulators, corticosterone, and schedule heightened aggression in mice /". Thesis, Connect to Dissertations & Theses @ Tufts University, 2003.
Cerca il testo completoAbstract (sommario):
Thesis (Ph. D.)--Tufts University, 2003.Advisers: Klaus Miczek; Joe DeBold. Submitted to the Dept. of Psychology. In title, GABAA is spelled GABA with a subscript A. Includes bibliographical references (leaves 146-183). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
46
Loftus, Katherine Marie. "Studies of the Structure and Function of E.coli Aspartate Transcarbamoylase". Thesis, Boston College, 2006. http://hdl.handle.net/2345/580.
Testo completoAbstract (sommario):
Thesis advisor: Evan R. KantrowitzE.coli Aspartate transcarbamoylase (ATCase) is the allosteric enzyme that catalyzes the committed step of the de novo pyrimidine biosynthesis pathway. ATCase facilitates the reaction between L-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate. The holoenzyme is a dodecamer, consisting of two trimers of catalytic chains, and three dimers of regulatory chains. ATCase is regulated homotropically by its substrates, and heterotropically by the nucleotides ATP, CTP, and UTP. These nucleotides bind to the regulatory chains, and alter the activity of the enzyme at the catalytic site. ATP activates the rate of ATCase's reaction, while CTP inhibits it. Additionally, UTP and CTP act together to inhibit the enzyme synergistically, each nucleotide enhancing the inhibitory effects of the other. Two classes of CTP binding sites have been observed, one class with a high affinity for CTP, and one with a low affinity. It has been theorized that the asymmetry of the binding sites is intrinsic to each of the three regulatory dimers. It has been hypothesized that the second observed class of CTP binding sites, are actually sites intended for UTP. To test this hypothesis, and to gain more information about heterotropic regulation of ATCase and signal transmission in allosteric enzymes, the construction of a hybrid regulatory dimer was proposed. In the successfully constructed hybrid, each of the three regulatory dimers in ATCase would contain one regulatory chain with compromised nucleotide binding. This project reports several attempts at constructing the proposed hybrid, but ultimately the hybrid enzyme was not attained. This project also reports preliminary work on the characterization of the catalytic chain mutant D141A. This residue is conserved in ATCase over a wide array of species, and thus was mutated in order to ascertain its significance
Thesis (BS) — Boston College, 2006
Submitted to: Boston College. College of Arts and Sciences
Discipline: Chemistry
Discipline: College Honors Program
47
Lampe, Jed N. "Allosteric mechanisms of cytochrome P450 3A4 probed using time-resolved fluorescence spectroscopy and steady-state kinetic analysis /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8164.
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Wohlever, Matthew L. "The role of the N domain in substrate binding, oligomerization, and allosteric regulation of the AAA+ Lon protease". Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/81034.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2013."June 2013." Cataloged from PDF version of thesis.
Includes bibliographical references.
For cells and organisms to survive, they must maintain protein homeostasis in varied and often harsh environments. Cells utilize proteases and chaperones to maintain their proteomes. In bacteria, most cytosolic proteolysis is performed by self-compartmentalized AAA+ proteases, which convert the chemical energy of ATP binding and hydrolysis into mechanical work to unfold and translocate substrates into an internal degradation chamber. Substrates are targeted to AAA+ proteases by degradation tags (degrons). In E. coli, the Lon protease is responsible for the degradation of numerous regulatory proteins, including the cell-division inhibitor SulA, but also recognizes and degrades the majority of misfolded proteins. How Lon recognizes and prioritizes such a vast array of substrates is poorly understood. Active Lon is a homohexamer in which each subunit contains an N domain, a AAA+ module that mediates ATP binding and hydrolysis, and a peptidase domain. Degron binding allosterically regulates Lon activity and can shift Lon into conformations with higher or lower protease activity, but the mechanistic basis of this regulation is unknown. The low-protease conformation of Lon may serve as a chaperone. In Chapter 2, I describe the development and characterization of fluorescent model substrates that Lon degrades in vitro and in vivo. In Chapter 3, I describe collaborative experiments that show that Lon equilibrates between a hexamer and a dodecamer. Based on biochemical analysis and a low-resolution EM dodecamer structure, Lon appears to shift its substrate profile by changing oligomeric states and contacts between N domains appear to stabilize the dodecamer. In Chapters 4 and 5, 1 identify a binding site for the sul20 degron (isolated from SulA) in the Lon N domain and demonstrate that substrate binding to this site allosterically regulates protease and ATPase activity. I also show that the E240K mutation in the N domain alters Lon activity and stabilizes dodecamers. Finally, I provide evidence that E. coli Lon can act as a chaperone in vivo. These experiments demonstrate that the N domain integrates substrate binding, oligomerization, and regulation of the catalytic activities of Lon.
by Matthew L. Wohlever.
Ph.D.
49
Parker, Mackenzie James. "Discovery and investigation of the novel overall activity allosteric regulation of the Bacillus subtilis class Ib ribonucleotide reductase". Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/109681.
Testo completoAbstract (sommario):
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.Page 490 blank. Cataloged from PDF version of thesis.
Includes bibliographical references.
Ribonucleotide reductases (RNRs) catalyze the reduction of nucleotides to 2'-deoxynucleotides in all organisms. Class lb RNRs consist of two subunits: a houses the catalytic and allosteric effector binding sites, and p houses a catalytically essential dimanganic-tyrosyl radical (Mn(III)2-Y*). The allosteric regulation of lb RNR activity has only been studied with the Salmonella enterica enzyme, which exhibits substrate specificity allosteric regulation by ATP and 2'-deoxynucleoside 5'-triphosphates (dNTPs), but not overall activity regulation by ATP and dATP. However, the S. enterica enzyme is not a good general model for Ib RNRs because it is not essential under most growth conditions, including pathogenesis. Other bacteria pathogenic to humans utilize lb RNRs as their sole source of dNTPs for DNA replication and repair. As RNR regulation plays a critical role in the high fidelity of these processes, the allosteric regulation of lb RNRs used as the primary dNTP supplier for a bacterium should be distinct from the S. enterica enzyme and, therefore, could provide a potential target for therapeutic development. Herein, the results of characterizing the allosteric regulation of the Ib RNR from the model organism Bacillus subtilis are presented. To facilitate these studies, we identified, cloned, and isolated the physiological reductant for RNR (thioredoxin/thioredoxin reductase/NADPH), thus allowing us to monitor activity spectrophotometrically. We discovered the effector dATP was a potent inhibitor of enzymatic activity at physiologically relevant concentrations, thereby demonstrating the first example of overall activity allosteric regulation in a class lb system. In other RNRs, overall activity regulation is mediated by a domain called the ATP-cone. This domain is absent from the B. subtilis enzyme; therefore, the inhibition represents a new mechanism of overall activity regulation. Analytical ultracentrifugation studies suggest dATP inhibition may be mediated by formation of large protein complexes. Biophysical studies also led to the discovery of tightly bound dAMP associated with a that increases the susceptibility of RNR to dATP inhibition. The potential physiological importance of dAMP is supported by studies examining YmaB, the unique fourth member of the B. subtilis RNR operon, which revealed this enzyme can hydrolyze dATP into dAMP and pyrophosphate and, therefore, might insert dAMP into a.
by Mackenzie James Parker.
Ph. D.
50
Beuter, Dominik [Verfasser], e Hannes [Akademischer Betreuer] Link. "Construction of Enzymes with Synthetic Allosteric Regulation to Control Metabolic Pathways of Escherichia coli / Dominik Beuter ; Betreuer: Hannes Link". Marburg : Philipps-Universität Marburg, 2019. http://d-nb.info/1193177480/34.
Testo completo