Unique Kinase Catalytic Mechanism of AceK with a Single Magnesium Ion

Abstract

Isocitrate dehydrogenase kinase/phosphatase (AceK) is the founding member of the protein phosphorylation system in prokaryotes. Based on the novel and unique structural characteristics of AceK recently uncovered, we sought to understand its kinase reaction mechanism, along with other features involved in the phosphotransfer process. Herein we report density functional theory QM calculations of the mechanism of the phosphotransfer reaction catalysed by AceK. The transition states located by the QM calculations indicate that the phosphorylation reaction, catalysed by AceK, follows a dissociative mechanism with Asp457 serving as the catalytic base to accept the proton delivered by the substrate. Our results also revealed that AceK prefers a single Mg2+-containing active site in the phosphotransfer reaction. The catalytic roles of conserved residues in the active site are discussed.

Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase

“…As shown in both Figs 1c and 3, Mg 2+ ion located at the center of the reaction system, 6-fold coordinated by E226, α-, β-, γ-phosphate groups and two water molecules throughout the reaction. This 6-fold coordination of Mg 2+ is essential for the phosphorylation reaction35. Our observation is in agreement with Hirano’s previous studies36.…”

How a Second Mg2+ Ion Affects the Phosphoryl-Transfer Mechanism in a Protein Kinase: A Computational Study

“…On the other hand, it is also interesting that some computational studies in other kinases have also shown that two Mg 2+ ions within the active site appear to be important for transition state stabilization and hence lowering of the activation energy, 48,54 while others have suggested that a second Mg 2+ ion would have a destabilizing effect on the transition state. 55,56 Therefore, a comprehensive study of the different structural motifs that could drive these observations is still needed.…”

Characterization of metal binding of bifunctional kinase/phosphatase AceK and implication in activity modulation

“…Previous calculations revealed that AceK preferred a single metal ion for the kinase activity and that a second metal ion would unfavorably increase the reaction energy barrier 29 . In comparison, the presence of a single metal ion would lead to a higher energy barrier pathway for the phosphatase activity 28 .…”

“…In light of the fact that only one metal was observed in AceK, it was hypothesized that AceK represents a novel type of phosphatase that distinguishes it from PPMs or PPPs; and the critical roles of the single Mg 2+ , ADP and Asp477 in the dephosphorylation were suggested 28 . Interestingly, recent theoretical studies showed that the presence of a single Mg 2+ ion would lead to a higher energy barrier pathway in the phosphatase reaction than the double Mg 2+ ions model, whereas the single metal model would lead to a lower energy barrier pathway in the kinase reaction 28,29 . This observation suggests that the metal ion in the catalytic center may have profound but opposing effects on kinase and phosphatase activities.…”