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

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 Ca 2+ 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 Ca 2+ cations with Mg 2+ 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.
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  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
OSTI Identifier:
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 25; Journal ID: ISSN 0021-9258
American Society for Biochemistry and Molecular Biology
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Laboratory Directed Research and Development (LDRD) Program
Country of Publication:
United States
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; crystal structure; enzyme mechanism; molecular dynamics; phosphoryl transfer; protein kinase