Phosphoryl Transfer Reaction Snapshots in Crystals: INSIGHTS INTO THE MECHANISM OF PROTEIN KINASE A CATALYTIC SUBUNIT

Background: PKAc (catalytic subunit) catalyzes phosphorylation of protein substrates thereby regulating a myriad of cellular processes. Results: X-ray structures of PKAc complexes along the phosphoryl transfer reaction have been obtained. Conclusion: The phosphotransfer follows a multistep mechanism, including conformational changes of the substrate and product groups, a loose transition state, and metal movement. Significance: Mechanistic knowledge about the phosphorylation by PKAc will contribute to understanding of the kinase function and regulation. 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. 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.