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

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.

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

10.1101/378133 ◽

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.

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

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180427150428 ◽

2018 ◽

Vol 18 (5) ◽

pp. 397-405 ◽

Cited By ~ 2

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.

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Fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli. Nature of bonds involved in the allosteric mechanism

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1990.tb19178.x ◽

1990 ◽

Vol 191 (3) ◽

pp. 701-704 ◽

Cited By ~ 12

Author(s):

Maria L. SPERANZA ◽

Giovanna VALENTINI ◽

Massimo MALCOVATI

Keyword(s):

Escherichia Coli ◽

Pyruvate Kinase ◽

Allosteric Mechanism ◽

Fructose 1,6 Bisphosphate

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Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro

Blood ◽

10.1182/blood.v64.3.607.bloodjournal643607 ◽

1984 ◽

Vol 64 (3) ◽

pp. 607-613 ◽

Cited By ~ 2

Author(s):

W Nijhof ◽

PK Wierenga ◽

GE Staal ◽

G Jansen

Keyword(s):

Pyruvate Kinase ◽

Gradient Centrifugation ◽

Recovery Period ◽

Erythroid Differentiation ◽

Glycolytic Enzyme ◽

Minor Amount ◽

Type I ◽

In Vitro Differentiation ◽

Percoll Density Gradient Centrifugation

Late committed progenitor cells of erythropoiesis, CFU-E (colony- forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure. The procedure included a four-day pretreatment of bled mice with the antibiotic thiamphenicol, a recovery period of 3 1/2 days, followed by centrifugal elutriation and Percoll density gradient centrifugation of the spleen cells. This practically pure CFU-E population was used to study some aspects of erythroid differentiation in vitro. Colony growth, as well as morphology and glycolytic enzyme activities of cells isolated at selected times of the 48-hour culture period, were determined. Marked declining activities of several enzymes, including hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase, and glucose-6- phosphate dehydrogenase, were observed during in vitro differentiation. The activity of diphosphoglycerate mutase was almost absent in the CFU- E, but progressively increased during differentiation. The isozyme distribution of aldolase and enolase did not change during CFU-E in vitro differentiation into the reticulocyte. Hexokinase (HK) in the CFU- E contained mainly a double-banded type I isozyme, in addition to a minor amount of HK II. During differentiation, a shift was noticed within the double-banded HK I region, whereas HK ii disappeared after one cell division. Pyruvate kinase in the CFU-E was characterized by the presence of both the K-type and the L-type isozyme and hybrids of these isozyme types. During in vitro differentiation, the production of the K-type isozyme rapidly stops in favor of the L type.

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In silicoidentification of putativeTrypanosoma cruzienolase inhibitors

10.1101/685206 ◽

2019 ◽

Author(s):

Edward A. Valera-Vera ◽

Melisa Sayé ◽

Chantal Reigada ◽

Mariana R. Miranda ◽

Claudio A. Pereira

Keyword(s):

Molecular Dynamics ◽

Molecular Docking ◽

Virtual Screening ◽

Active Site ◽

Screening Strategy ◽

Glycolytic Enzyme ◽

Docking Simulations ◽

Enzyme Active Site ◽

Key Enzyme

AbstractEnolase is a glycolytic enzyme that catalyzes the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. In trypanosomatids enolase was proposed as a key enzyme afterin silicoandin vivoanalysis and it was validated as a protein essential for the survival of the parasite. Therefore, enolase constitutes an interesting enzyme target for the identification of drugs against Chagas disease. In this work, a combined virtual screening strategy was implemented, employing similarity virtual screening, molecular docking and molecular dynamics. First, two known enolase inhibitors and the enzyme substrates were used as queries for the similarity screening on the Sweetlead database using five different algorithms. Compounds retrieved in the top 10 of at least three search algorithms were selected for further analysis, resulting in six compounds of medical use (etidronate, pamidronate, fosfomycin, acetohydroximate, triclofos, and aminohydroxybutyrate). Molecular docking simulations predicted acetohydroxamate and triclofos would not bind to the active site of the enzyme, and a re-scoring of the obtained poses signaled fosfomycin and aminohydroxybutyrate as bad enzyme binders. Docking poses obtained for etidronate, pamidronate, and PEP, were used for molecular dynamics calculations to describe their mode of binding. From the obtained results, we propose etidronate as a possibleTcENO inhibitor, and describe desirable and undesirable molecular motifs to be taken into account in the repurposing or design of drugs aiming this enzyme active site.

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The effect of enthalpy of mixing on the atomic level structure and plasticity of amorphous alloys: A molecular dynamics simulation study in a binary model system

Intermetallics ◽

10.1016/j.intermet.2017.09.006 ◽

2018 ◽

Vol 92 ◽

pp. 25-35 ◽

Cited By ~ 3

Author(s):

Y.S. Yun ◽

B.J. Kim ◽

J.H. Na ◽

H.-S. Nam ◽

P.-R. Cha ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Simulation Study ◽

Amorphous Alloys ◽

Level Structure ◽

Enthalpy Of Mixing ◽

Model System ◽

Atomic Level ◽

Dynamics Simulation ◽

Binary Model

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Pyruvate Kinase (PK) Deficiency Hereditary Nonspherocytic Hemolytic Anemia

Blood ◽

10.1182/blood.v19.3.267.267 ◽

1962 ◽

Vol 19 (3) ◽

pp. 267-295 ◽

Cited By ~ 169

Author(s):

KOUICHI R. TANAKA ◽

WILLIAM N. VALENTINE ◽

SHIRO MIWA

Keyword(s):

Pyruvate Kinase ◽

Hemolytic Anemia ◽

Glycolytic Enzyme ◽

Enzyme Deficiency ◽

Exact Nature ◽

Symptomatic Disease ◽

Genetically Determined ◽

Recessive Transmission ◽

Enzymatic Defect

Abstract 1. The erythrocytes of seven patients conforming to the criteria of Type II congenital nonspherocytic hemolytic anemia have been demonstrated to have a specific deficiency in the glycolytic enzyme pyruvate kinase. Other glycolytic enzymes, glucose-6-phosphate and 6-phosphogluconic dehydrogenases, and certain non-glycolytic erythrocyte enzymes are normally active. The leukocytes of these patients possess normal pyruvate kinase activity. 2. Although no inhibitors were detected, the exact nature of the enzymatic defect remains to be elucidated. 3. Family studies provide strong evidence for a genetically determined disorder and are consistent with an autosomal recessive transmission of the defect. A partial enzyme deficiency, not reflected in clinical disease, is present in heterozygotes. The symptomatic disease, though variable in severity, appears to be due to homozygosity for the defect. 4. It is suggested that the enzyme deficiency is pathogenetically related to the premature demise of the red cells in vivo. 5. The name "pyruvate kinase (PK) deficiency hereditary nonspherocytic hemolytic anemia" is proposed for these patients.

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Biochemical and structural insights into how amino acids regulate pyruvate kinase muscle isoform 2

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013030 ◽

2020 ◽

Vol 295 (16) ◽

pp. 5390-5403 ◽

Cited By ~ 1

Author(s):

Suparno Nandi ◽

Mishtu Dey

Keyword(s):

Pyruvate Kinase ◽

Cancer Metabolism ◽

Gel Filtration ◽

Binding Pocket ◽

Glycolytic Enzyme ◽

X Ray Crystallography ◽

Highly Active ◽

Attractive Therapeutic Target ◽

Fluorescence Binding ◽

Small Molecule Modulators

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

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Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro

Blood ◽

10.1182/blood.v64.3.607.607 ◽

1984 ◽

Vol 64 (3) ◽

pp. 607-613 ◽

Cited By ~ 23

Author(s):

W Nijhof ◽

PK Wierenga ◽

GE Staal ◽

G Jansen

Keyword(s):

Pyruvate Kinase ◽

Gradient Centrifugation ◽

Recovery Period ◽

Erythroid Differentiation ◽

Glycolytic Enzyme ◽

Minor Amount ◽

Type I ◽

In Vitro Differentiation ◽

Percoll Density Gradient Centrifugation

Abstract Late committed progenitor cells of erythropoiesis, CFU-E (colony- forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure. The procedure included a four-day pretreatment of bled mice with the antibiotic thiamphenicol, a recovery period of 3 1/2 days, followed by centrifugal elutriation and Percoll density gradient centrifugation of the spleen cells. This practically pure CFU-E population was used to study some aspects of erythroid differentiation in vitro. Colony growth, as well as morphology and glycolytic enzyme activities of cells isolated at selected times of the 48-hour culture period, were determined. Marked declining activities of several enzymes, including hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase, and glucose-6- phosphate dehydrogenase, were observed during in vitro differentiation. The activity of diphosphoglycerate mutase was almost absent in the CFU- E, but progressively increased during differentiation. The isozyme distribution of aldolase and enolase did not change during CFU-E in vitro differentiation into the reticulocyte. Hexokinase (HK) in the CFU- E contained mainly a double-banded type I isozyme, in addition to a minor amount of HK II. During differentiation, a shift was noticed within the double-banded HK I region, whereas HK ii disappeared after one cell division. Pyruvate kinase in the CFU-E was characterized by the presence of both the K-type and the L-type isozyme and hybrids of these isozyme types. During in vitro differentiation, the production of the K-type isozyme rapidly stops in favor of the L type.

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