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

Abstract

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.

Phosphoryl Transfer Reaction Snapshots in Crystals

“…In addition, we recently obtained an x-ray structure of the PKAc-Ca 2 ADP-pSP20 complex, in which the ADP and phosphorylated SP20 products were captured in the active site (8). Comparison of our current pseudo-Michaelis complex with the transition state mimic and the product allows us to visualize structural changes that may accompany the catalysis and to identify mechanistically important structural information.…”

“…The structure is consistent with the S N 2 mechanism and demonstrates a short interaction between Asp-166 and the side chain of Ser-21 SP20 . 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).…”

“…PKAc requires one or two divalent metal ions to bind to the active site to be active (5,6). The physiological metal is magnesium, although others can support phosphotransferase activity (7,8).…”

“…In particular, IP20 lacks the nucleophilic group, having Ala in place of reactive Ser, and precluding the analysis of the conformation and interactions of the nucleophile before the reaction (15,25,26). In addition, in complexes containing substrate peptide SP20 and ATP analogs, the side chain of Ser-21 SP20 adopts a conformation, in which it is rotated away from Asp-166, which prevents H-bond formation (8,27). The transition state mimic, having the MgF 3 Ϫ anion in place of ␥-PO 3 , ADP, and SP20 has also been determined (23).…”

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

“…Interestingly, the Mg 2+ to Ca 2+ substitution effects are typically also similarly inhibitory 76 for two-metal ion catalysis as well as for the one-metal ion catalysis in dUTPase [21][22] and in several ATPases [77][78][79][80][81] and kinases 78,[82][83] with some notable exceptions. [84][85] This key metal ion often coordinates the cleaved phosphate. This helps the catalysis by lowering the pK a of the phosphate group.…”