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A Quantitative Comparison of Wild‐Type and Gatekeeper Mutant Cdk2 for Chemical Genetic Studies with ATP Analogues
Author(s) -
Elphick Lucy M.,
Lee Sarah E.,
Child Emma S.,
Prasad Aarathi,
Pignocchi Cristina,
Thibaudeau Sébastien,
Anderson Alexandra A.,
Bonnac Laurent,
Gouverneur Véronique,
Mann David J.
Publication year - 2009
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900052
Subject(s) - cyclin dependent kinase 2 , mutant , kinase , cyclin dependent kinase , biochemistry , biology , phosphorylation , substrate level phosphorylation , substrate (aquarium) , wild type , microbiology and biotechnology , chemistry , protein kinase a , cell cycle , gene , ecology
Mutant kinase kinetics : Protein kinases with enlarged ATP binding sites are increasingly being used as tools to probe the functioning signal transduction cascades. Using human cyclin‐dependent kinase 2 as a model system, we demonstrate that enlargement of the ATP binding site does not substantially alter either the catalysis kinetics nor substrate or phosphorylation site selection.Chemical genetic studies with enlarged ATP binding sites and unnatural ATP analogues have been applied to protein kinases for characterisation and substrate identification. Although this system is becoming widely used, there are limited data available about the kinetic profile of the modified system. Here we describe a detailed comparison of the wild‐type cdk2 and the mutant gatekeeper kinase to assess the relative efficiencies of these kinases with ATP and unnatural ATP analogues. Our data demonstrate that mutation of the kinase alters neither the substrate specificity nor the phosphorylation site specificity. We find comparable K M /V max values for mutant cdk2 and wild‐type kinase. Furthermore, F80G cdk2 is efficiently able to compensate for a defective cdk in a biological setting.