TY - JOUR
T1 - Phosphoryl transfer reaction snapshots in crystals
T2 - Insights into the mechanism of protein kinase a catalytic subunit
AU - Gerlits, Oksana
AU - Tian, Jianhui
AU - Das, Amit
AU - Langan, Paul
AU - Heller, William T.
AU - Kovalevsky, Andrey
N1 - Publisher Copyright:
© 2015, American Society for Biochemistry and Molecular Biology Inc. All rights reserved.
PY - 2015/6/19
Y1 - 2015/6/19
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84938711523&partnerID=8YFLogxK
U2 - 10.1074/jbc.M115.643213
DO - 10.1074/jbc.M115.643213
M3 - Article
C2 - 25925954
AN - SCOPUS:84938711523
SN - 0021-9258
VL - 290
SP - 15538
EP - 15548
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 25
ER -