Abstract
BackgroundIt has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings.ResultsWe have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP.ConclusionsThe data outlined serves to demonstrate the “push” mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved “push” and “pull” mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the γ-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.
Highlights
It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes
The adenylate kinases have two nucleotide binding sites and the equivalent amino acid residues in the second adenylate kinase 1 (AK1) binding site are carbonyl associated with the C6-NH2, the Thr associated with the proton transfer from C8H to the α-PO4 (Thr 39), the Arg associated with C8 protonation (Arg97), the Arg coordinated to the α-PO4 and β-PO4 (Arg44/138), Lys associated with the γ-PO4 protonation (Lys21)
The mutations carried out on both shikimate kinase (SK) and AK1 were: the Thr associated with the proton transfer from C8H to the α-PO4 (SK, Thr17; AK1, Thr 23 and Thr 39), the Arg associated with C8 protonation (SK, Arg110: AK1; Arg128 and Arg97), the Arg co-ordinated to the α-PO4 and β-PO4 (SK, Arg117; AK1, Arg132), and the Lys associated with the γ-PO4 protonation (SK, Lys15)
Summary
It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. All the kinase mechanistic classes were clustered into two proposed mechanisms depending on how the C8-H is induced to be labile, namely by either the co-ordination of a backbone carbonyl to C6-NH2 of the adenyl moiety (a “push” mechanism), or based on the protonation of N7 of the adenyl moiety (a “pull” mechanism) 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 Group 2 kinases are based on the kinase organization outlined by Cheek et al [3])
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