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

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.

Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies

“…The specific activity of hAK1 and AKst in the direction of ATP synthesis (1 mM ADP as a substrate) was 87 ± 1 and 323 ± 4 U/mg of protein, respectively, whereas in the direction of ADP synthesis (AMP and ATP as substrates, 1 mM each) the activity was 463 ± 14 and 1100.6 ± 20 U/mg of protein, respectively. The specific activities of the recombinant enzymes were similar to that reported by other authors [ 43 , 57 , 58 , 59 ]. The CD spectrum and the calculated content of secondary structure showed that both recombinant AKs are predominately helical but with the significant content of β-sheet ( Figure S1B ).…”

Emerging Enzymes for ATP Regeneration in Biocatalytic Processes

“…Additional functions include its role as a signalling molecule for posttranslational modification and catalytic functions within enzymes (Scheme ) 3. 5 From this large diversity of functions it becomes clear that describing ATP solely as energy currency in biochemistry is inadequate 6. ATP, therefore, is involved in many levels of biological function (Figure 1).…”

“…As described above, one important issue in applying cofactor‐dependent enzymes is cofactor regeneration. A range of efficient regeneration systems have been successfully developed for many organic enzyme cofactors, such as NAD(P)H 5. 31, 32 A key parameter for cofactor‐dependent enzymes within this context is the total turnover number (TTN), which indicates the moles of product formed per mole of cofactor during its entire life and represents a measure for catalytic efficacy.…”