Direct Kinetic Evidence That Lysine 215 Is Involved in the Phospho-Transfer Step of Human 3-Phosphoglycerate Kinase
Autor: | Tom Barman, Perrine Lallemand, Nancy Adamek, Christian Valentin, Andrea Varga, Laurent Chaloin, Mária Vas, Judit Szabó, Corinne Lionne |
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Přispěvatelé: | Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Department of Physical Geography |
Rok vydání: | 2009 |
Předmět: |
Models
Molecular Mutant MESH: Catalytic Domain Biochemistry Phosphotransferase 03 medical and health sciences Catalytic Domain Humans MESH: Lysine [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Nucleotide 030304 developmental biology chemistry.chemical_classification 0303 health sciences Phosphoglycerate kinase MESH: Humans MESH: Kinetics biology Kinase Lysine 030302 biochemistry & molecular biology Active site Kinetics Phosphoglycerate Kinase Enzyme chemistry biology.protein MESH: Phosphoglycerate Kinase Salt bridge MESH: Models Molecular |
Zdroj: | Biochemistry Biochemistry, American Chemical Society, 2009, 48 (29), pp.6998-7008. ⟨10.1021/bi900396h⟩ |
ISSN: | 1520-4995 0006-2960 |
Popis: | International audience; 3-Phosphoglycerate kinase (PGK) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. PGK is a key enzyme in glycolysis; it catalyzes the reversible reaction 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP. Here we explored the catalytic role in human PGK of the highly conserved Lys 215 that has been proposed to be essential for PGK function by a transient and equilibrium kinetic study with the active site mutant K215A. By the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type PGK and mutant K215A. By the use of a chemical sampling method (rapid quench flow) under multiple and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild-type PGK follows product formation (presumably product release), whereas with the mutant it is the phospho-transfer step itself that is rate-limiting. Mutant K215A has a low inherent phosphotransferase activity, and to explain this, we carried out a molecular modeling study. This suggests that with the mutant the conserved Arg 65 replaces the missing Lys 215 by helping to position the transferable phospho group during the reaction. Molecular dynamics simulations suggest that in the mutant the closed conformation of the enzyme is stabilized by a salt bridge between Asp 218 and Arg 170 rather than Arg 65 in the wild-type PGK. |
Databáze: | OpenAIRE |
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