title: Kinetic and mechanistic characterisation of Choline Kinase-α.
abstract: Choline Kinase is a key component of the Kennedy pathway that converts choline into a number of structural and signalling lipids that are essential for cell growth and survival. One member of the family, Choline Kinase-α (ChoKα) is frequently up-regulated in human cancers, and expression of ChoKα is sufficient to transform cells. Consequently ChoKα has been studied as a potential target for therapeutic agents in cancer research. Despite great interest in the enzyme, mechanistic studies have not been reported. In this study, a combination of initial velocity and product inhibition studies, together with the kinetic and structural characterisation of a novel ChoKα inhibitor is used to support a mechanism of action for human ChoKα. Substrate and inhibition kinetics are consistent with an iso double displacement mechanism, in which the γ-phosphate from ATP is transferred to choline in two distinct steps via a phospho-enzyme intermediate. Co-crystal structures, and existing site-specific mutation studies, support an important role for Asp306, in stabilising the phospho-enzyme intermediate. The kinetics also indicate a distinct kinetic (isomerisation) step associated with product release, which may be attributed to a conformational change in the protein to disrupt an interaction between Asp306 and the phosphocholine product, facilitating product release. This study describes a mechanism for ChoKα that is unusual amongst kinases, and highlights the availability of different enzyme states that can be exploited for drug discovery.
authors: Vertex Pharmaceuticals (Europe) Limited, Oxfordshire, UK.
authors: Catherine S
authors: CS
authors: Hudson
authors: Ronald M
authors: RM
authors: Knegtel
authors: Kieron
authors: K
authors: Brown
authors: Peter A
authors: PA
authors: Charlton
authors: John R
authors: JR
authors: Pollard
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D002797:Choline Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003460:Crystallization; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010767:Phosphorylcholine / Q000378:metabolism
chemical_list: D010767:Phosphorylcholine; D000255:Adenosine Triphosphate; C517212:CHKA protein, human; D002797:Choline Kinase