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Title: Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

Journal Article · · Journal of Biological Chemistry
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  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
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To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

Research Organization:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Organization:
USDOE Laboratory Directed Research and Development (LDRD) Program
Grant/Contract Number:
AC05-00OR22725
OSTI ID:
1201297
Journal Information:
Journal of Biological Chemistry, Vol. 290, Issue 25; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 19 works
Citation information provided by
Web of Science

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Cited By (5)

Molecular Fingerprints for a Novel Enzyme Family in Actinobacteria with Glucosamine Kinase Activity journal May 2019
Zooming in on protons: Neutron structure of protein kinase A trapped in a product complex journal March 2019
Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes journal March 2017
Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα journal August 2017
Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes book March 2017

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
crystal structure
enzyme mechanism
molecular dynamics
phosphoryl transfer
protein kinase