title: Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.
abstract: The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.
authors.affiliation: Chemistry Department, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Sanela
authors.initials: S
authors.lastname: Martić
authors.forename: Meghan K
authors.initials: MK
authors.lastname: Rains
authors.forename: Daniel
authors.initials: D
authors.lastname: Freeman
authors.forename: Heinz-Bernhard
authors.initials: HB
authors.lastname: Kraatz
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D053148:Caspase 3; D002384:Catalysis; D005296:Ferrous Compounds; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D000075163:Metallocenes; D015336:Molecular Probe Techniques; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005296:Ferrous Compounds; D000075163:Metallocenes; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D053148:Caspase 3; C004998:ferrocene