Protein kinase A in the neutron beam: Insights for catalysis from directly observing protons.
Enzyme mechanism
Hydrogen bond
Neutron crystallography
Phosphoryl transfer
Product complex
Protein kinase A
Proton transfer
Protonation state
Journal
Methods in enzymology
ISSN: 1557-7988
Titre abrégé: Methods Enzymol
Pays: United States
ID NLM: 0212271
Informations de publication
Date de publication:
2020
2020
Historique:
entrez:
26
2
2020
pubmed:
26
2
2020
medline:
24
6
2021
Statut:
ppublish
Résumé
Protein kinases transmit chemical signals by phosphorylating substrate proteins, thus regulating a multitude of cellular processes. cAMP-dependent protein kinase (PKA), a prototypical enzyme for the whole kinase family, has been the focus of research for several decades, however, the details of the chemical mechanism of phosphoryl group transfer have remained unknown. We used neutron crystallography to map key proton sites and hydrogen bonding interactions in the PKA catalytic subunit (PKAc) in a product complex containing adenosine diphosphate (ADP) and the phosphorylated high affinity protein kinase substrate (pPKS) peptide. To improve neutron diffraction, we deuterated PKAc allowing us to use very small crystals. In the product complex, the phosphoryl group of pPKS is protonated whereas the catalytic Asp166 is not. H/D exchange analysis of the main-chain amides and comparison with the NMR analysis of PKAc with inhibitor peptide complex revealed exchangeable amides that may distinguish the catalytic and inhibited states.
Identifiants
pubmed: 32093838
pii: S0076-6879(19)30480-X
doi: 10.1016/bs.mie.2019.12.003
pmc: PMC7494206
mid: NIHMS1626525
pii:
doi:
Substances chimiques
Protons
0
Cyclic AMP-Dependent Protein Kinases
EC 2.7.11.11
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
311-331Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM019301
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM100310
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM130389
Pays : United States
Organisme : NIGMS NIH HHS
ID : R37 GM019301
Pays : United States
Informations de copyright
© 2020 Elsevier Inc. All rights reserved.
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