Eukaryotic protein kinases catalyze the phosphorylation of their substrate proteins on serine, threonine or tyrosine residues using adenosine triphosphate (ATP) as the donor of the phosphate group. As a major mode of posttranslational modification, phosphorylation by protein kinases plays a pivotal role in transducing signals and regulating numerous cellular processes such as growth, proliferation, differentiation, metabolism and motility. Although extensive biochemical and structural characterizations of protein kinases have been carried out, the details behind the catalytic mechanism of protein kinases are not clear. We sought to investigate the role of the a-� bridging oxygen in kinase catalysis through the use of unnatural ATP analogs. A new method featuring the use of a chiral auxiliary group was established to afford individual diastereomers of a,�-CHX-ATP (X = F or Cl). By using various assays such as real-time NMR spectroscopy and a fluorescence-based kinase assay, we demonstrate that a tyrosine kinase Src exhibits high diastereoselectivity utilizing a,�-CHX-ATP. The co-crystal structure of Src and a,�-CHF-ADP, the product of the superior nucleotide substrate, was solved to establish the absolute stereoconfiguration of the CHF carbon and to elucidate molecular basis of the exquisite diastereoselectivity exhibited by the enzyme. The crystal structures reveal the presence of a hydrogen bond between the fluorine atom and the amino group of the catalytic lysine, which appears to be important for the nucleotide to assume a catalytically competent conformation. We conclude that the similar interaction between the a-� bridging oxygen atom of ATP and the lysine is important for efficient catalysis by protein kinases.

Diastereoselective utilization of novel ATP analogs by a protein kinase Src

248th National Meeting (2014)

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