Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer

Abstract

BackgroundThe kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes.ResultsA number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile.ConclusionsThe "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.

The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

“…It has been demonstrated that the adenyl group of ATP plays a direct role in the control of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes [1,2]. The role of the ATP C8-H in the binding and/or phosphoryl transfer activity of a number of kinase and synthetase enzymes was elucidated in comparative enzyme activity assays using ATP and ATP deuterated at the C8 position.…”

“…The functionality required for the catalysis and regulation of phosphoryl transfer was found to be conserved within the families or fold-groups. As a result, a number of conserved mechanisms were proposed, wherein the C8-H of the adenyl moiety was found to play a direct role in the control of phosphoryl transfer [2]. These mechanisms were developed using structurally conserved amino acid residues within hydrogen-bonding distance of a nucleotide in the active sites of crystallised kinases of a particular mechanistic class.…”

“…Associated with both the “push” and “pull” mechanisms is a proton transfer cascade via the tri-phosphate backbone, initiated from C8-H, and mediated by specific conserved amino acid residues unique to a particular mechanistic “class” of kinases, that culminates in a pentavalent species formed between the γ-phosphate of ATP and the substrate nucleophile. The “push” mechanism was defined within the Group 2 kinases [2]. The Group 2 kinases are based on the kinase organization outlined by Cheek et al [3]).…”

Structure of the acetophenone carboxylase core complex: prototype of a new class of ATP-dependent carboxylases/hydrolases

“…The known ATP binding sites in kinases also define the approximate substrate positions in the vicinity of the γ-phosphates of ATP26. In Apcα, the potential substrate binding site resembles similarly-sized pockets in pantothenate or acetate kinases2327 (Fig.…”

Theoretical Studies of the Catalytic Mechanism of the Dihydroxyacetone Kinase

“…34,35 These enzymes normally hold two Mg 2+ ions placed into the active site which assist the phosphorylation catalysis (the role of Mg 2+ ions will be discussed in the section 4.2).…”