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

Abstract

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 apo-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.

Phosphoryl Transfer Reaction Snapshots in Crystals

“…Additionally, the side chain of Cys-21 of CP20 clearly displays two conformations as indicated by the omit difference electron density map, which would not be possible with 100% PO 4 retention. Conformation A (64% occupancy), in which the C␤-S␥ bond rotated toward the bulk solvent and away from Asp-166, is similar to that observed in all product complexes (8,14), and in PKAc-Ca 2 AMPPNP-SP20 complex. Conformation B (36% occupancy) with the SH group pointing toward Asp-166 is identical to the position of C␤-S␥ in PKAc-Ca 2 ATP-CP20 and clashing into the free phosphate (Fig.…”

“…Finally, PKAc product complexes have been trapped in crystals when ATP and SP20 were utilized (8). These structures, and enzyme kinetics measurements, demonstrated that, in addition to Mg 2ϩ , all other divalent alkaline earth metals, including Ca 2ϩ , Sr 2ϩ , and Ba 2ϩ , support the phosphoryl transfer to SP20 (14). In our preliminary studies, the rate of phosphotransfer determined by pre-steady state kinetics for RII␤ holoenzyme in the presence of Ca 2ϩ is only severalfold lower than with Mg 2ϩ .…”

“…Complexes of PKAc with nucleotide and/or substrate analogs are found in three major conformational states that differ in the relative orientation of the N-and C-lobes. With no ligands bound (apo form) PKAc adopts an open conformation; upon nucleotide or substrate binding (binary form), PKAc transitions to an intermediate, partially closed, state; last, PKAc assumes a closed conformation when all components for the reaction are in place (ternary form) (12)(13)(14)(15).…”

“…In PKAc, Asp-166 is the nearest ionizable residue to the OH group of the substrate and may act as a catalytic base and/or to correctly position the nucleophile. Asp-166 is universally conserved in the active site of all protein kinases and can form hydrogen bond interactions with Ser of the substrate (14,22,23). Asp-to-Ala substitution produces a mutant variant with activity below 1% of the wild-type (24).…”

Divalent Metal Ions Mg2+ and Ca2+ Have Distinct Effects on Protein Kinase A Activity and Regulation

“…20 We tested whether the PKA catalytic subunit (human isoform C α 1) could also phosphorylate a real protein substrate in the presence of calcium. To examine this, we generated a protein substrate, termed PKS, where only the P0 site Ala of full-length PKI is replaced by Ser (see Figure 1).…”