Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X‑ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20

X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg²⁺, nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5′-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues

Metal-Free cAMP-Dependent Protein Kinase Can Catalyze Phosphoryl Transfer

X-ray structures of several ternary product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with no bound metal ions and with Na⁺ or K⁺ coordinated at two metal-binding sites. The metal-free PKAc and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. In all studied complexes, the ATP and the substrate peptide (SP20) were modified into the products ADP and the phosphorylated peptide. The products of the phosphotransfer reaction were also found when ATP-γS, a nonhydrolyzable ATP analogue, reacted with SP20 in the PKAc active site containing no metals. Single turnover enzyme kinetics measurements utilizing ³²P-labeled ATP confirmed the phosphotransferase activity of the enzyme in the absence of metal ions and in the presence of alkali metals. In addition, the structure of the <i>apo</i>-PKAc binary complex with SP20 suggests that the sequence of binding events may become ordered in a metal-free environment, with SP20 binding first to prime the enzyme for subsequent ATP binding. Comparison of these structures reveals conformational and hydrogen bonding changes that might be important for the mechanism of catalysis