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Protein kinase A in the neutron beam: Insights for catalysis from directly observing protons

Journal Article · · Methods in Enzymology
 [1];  [2];  [3];  [4];  [5];  [2]
  1. Tennessee Wesleyan Univ., Athens, TN (United Staes)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Inst. Laue-Langevin (ILL), Grenoble (France)
  4. Univ. of Minnesota, Minneapolis, MN (United States)
  5. Trinium Research, Inc., San Diego, CA (United States)
+ Show Author Affiliations

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.

Research Organization:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Organization:
USDOE
Grant/Contract Number:
AC05-00OR22725
OSTI ID:
1606040
Journal Information:
Methods in Enzymology, Journal Name: Methods in Enzymology Journal Issue: 1 Vol. 634; ISSN 0076-6879
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Enzyme mechanism
Hydrogen bond
Neutron crystallography
Phosphoryl transfer
Product complex
Protein kinase A
Proton transfer
Protonation state