The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases.

Colin P Kenyon,Paul A Steenkamp,Thokozani C Nkosi,Anjo Steyn,Robyn L Roth,Lyndon C Oldfield

doi:10.1186/1471-2091-12-36

Colin P Kenyon, Paul A Steenkamp + Show 4 more

Open Access

https://doi.org/10.1186/1471-2091-12-36

Copy DOI

Abstract

BackgroundThe kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude.ResultsWe have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold.ConclusionsKinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of Km associated with kinase enzymes.

Highlights

The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer
Both prokaryotic and eukaryotic organisms are represented with kinase isoenzymes that appear to be kinetically and functionally distinct based on the rate of phosphoryl transfer and the regulation thereof. This investigation was undertaken to ascertain the extent to which the adenyl group within ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. To this end the role of the C8-H of ATP on the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes was elucidated in comparative enzyme activity essays using ATP and ATP deuterated at the C8 position
Where possible the effect of the ATP and C8D-ATP on the specific activity of the enzyme was expressed over a concentration profile that included the ATP or C8D-ATP concentrations that tended towards vmax as well as an ATP or C8D-ATP concentration profile at low concentrations that would allow for the accurate determination of the Kinetic isotope effects (KIE)

Summary

Introduction

The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The ligases are enzymes catalysing the joining of two molecules with the concomitant hydrolysis of single group, both prokaryotic and eukaryotic organisms are represented with kinase isoenzymes that appear to be kinetically and functionally distinct based on the rate of phosphoryl transfer and the regulation thereof. This investigation was undertaken to ascertain the extent to which the adenyl group within ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. Stearic effects affect the KIE to the same extent as the secondary KIE’s

Methods

Results

Discussion

Conclusion