scholarly journals Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

2018 ◽  
Author(s):  
Jamie A. Macpherson ◽  
Alina Theisen ◽  
Laura Masino ◽  
Louise Fets ◽  
Paul C. Driscoll ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
High Activity ◽  
Pyruvate Kinase ◽  
Cross Talk ◽  
Glycolytic Enzyme ◽  
Computational Framework ◽  
Allosteric Inhibitor ◽  
Fructose 1,6 Bisphosphate ◽  
Low Activity

ABSTRACTAllosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

scholarly journals Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jamie A Macpherson ◽  
Alina Theisen ◽  
Laura Masino ◽  
Louise Fets ◽  
Paul C Driscoll ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Mutual Information ◽  
Enzymatic Activity ◽  
Pyruvate Kinase ◽  
Glycolytic Enzyme ◽  
Model System ◽  
Eigenvalue Decomposition ◽  
Coordinate Regulation ◽  
Maximal Activation ◽  
Fructose 1,6 Bisphosphate

Several enzymes can simultaneously interact with multiple intracellular metabolites, however, how the allosteric effects of distinct ligands are integrated to coordinately control enzymatic activity remains poorly understood. We addressed this question using, as a model system, the glycolytic enzyme pyruvate kinase M2 (PKM2). We show that the PKM2 activator fructose 1,6-bisphosphate (FBP) alone promotes tetramerisation and increases PKM2 activity, but addition of the inhibitor L-phenylalanine (Phe) prevents maximal activation of FBP-bound PKM2 tetramers. We developed a method, AlloHubMat, that uses eigenvalue decomposition of mutual information derived from molecular dynamics trajectories to identify residues that mediate FBP-induced allostery. Experimental mutagenesis of these residues identified PKM2 variants in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings reveal residues involved in FBP-induced allostery that enable the integration of allosteric input from Phe and provide a paradigm for the coordinate regulation of enzymatic activity by simultaneous allosteric inputs.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

The Role of Sensing Peptides in the Cross-talk between Microbiota and Human Cancer Cells

2018 ◽  
Vol 18 (18) ◽  
pp. 1567-1571
Author(s):  
Anna Lucia Tornesello ◽  
Luigi Buonaguro ◽  
Maria Lina Tornesello ◽  
Franco M. Buonaguro
Keyword(s):  
Cancer Cells ◽  
Cross Talk ◽  
Human Cancer ◽  
Human Cancer Cells ◽  
The Cross

Structure-Based Virtual Screening and Biochemical Evaluation for the Identification of Novel Trypanosoma brucei Aldolase Inhibitors

2018 ◽  
Vol 18 (5) ◽  
pp. 397-405 ◽  
Author(s):  
Leonardo L.G. Ferreira ◽  
Rafaela S. Ferreira ◽  
David L. Palomino ◽  
Adriano D. Andricopulo
Keyword(s):  
Drug Discovery ◽  
Virtual Screening ◽  
Trypanosoma Brucei ◽  
Enzyme Inhibitor ◽  
Practical Knowledge ◽  
Glycolytic Enzyme ◽  
Structure Based Drug Design ◽  
Biochemical Evaluation ◽  
Dynamics Simulations ◽  
Fructose 1,6 Bisphosphate

Introduction: The glycolytic enzyme fructose-1,6-bisphosphate aldolase is a validated molecular target in human African trypanosomiasis (HAT) drug discovery, a neglected tropical disease (NTD) caused by the protozoan Trypanosoma brucei. Herein, a structure-based virtual screening (SBVS) approach to the identification of novel T. brucei aldolase inhibitors is described. Distinct molecular docking algorithms were used to screen more than 500,000 compounds against the X-ray structure of the enzyme. This SBVS strategy led to the selection of a series of molecules which were evaluated for their activity on recombinant T. brucei aldolase. The effort led to the discovery of structurally new ligands able to inhibit the catalytic activity of the enzyme. Results: The predicted binding conformations were additionally investigated in molecular dynamics simulations, which provided useful insights into the enzyme-inhibitor intermolecular interactions. Conclusion: The molecular modeling results along with the enzyme inhibition data generated practical knowledge to be explored in further structure-based drug design efforts in HAT drug discovery.

scholarly journals Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 216
Author(s):  
Alanna C. Tseng ◽  
Vivek R. Nerurkar ◽  
Kabi R. Neupane ◽  
Helmut Kae ◽  
Pakieli H. Kaufusi
Keyword(s):  
West Nile Virus ◽  
Enzymatic Activity ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
West Nile ◽  
Biochemical Assays ◽  
In Trans ◽  
Viral Polyprotein ◽  
Ns3 Protease

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

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