10.1021/bi5000965.s001 Oksana Gerlits Oksana Gerlits Amit Das Amit Das Malik M. Keshwani Malik M. Keshwani Susan Taylor Susan Taylor Mary Jo Waltman Mary Jo Waltman Paul Langan Paul Langan William T. Heller William T. Heller Andrey Kovalevsky Andrey Kovalevsky Metal-Free cAMP-Dependent Protein Kinase Can Catalyze Phosphoryl Transfer American Chemical Society 2015 phosphoryl transfer reaction nonhydrolyzable ATP analogue ternary product complexes SP 20 binding peptide ADP metal ions site SP 20 alkali metals PKAc Single turnover enzyme kinetics measurements 2015-12-17 02:14:07 Journal contribution https://acs.figshare.com/articles/journal_contribution/Metal_Free_cAMP_Dependent_Protein_Kinase_Can_Catalyze_Phosphoryl_Transfer/2033409 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<sup>+</sup> or K<sup>+</sup> 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 <sup>32</sup>P-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.