Details

Autor(en) / Beteiligte

• Hudson, Catherine S.
• Knegtel, Ronald M.
• Brown, Kieron
• Charlton, Peter A.
• Pollard, John R.

Titel

Kinetic and mechanistic characterisation of Choline Kinase-α

Ist Teil von

• Biochimica et biophysica acta, 2013-06, Vol.1834 (6), p.1107-1116

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2013

Quelle

Access via ScienceDirect (Elsevier)

Beschreibungen/Notizen

• 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. •An iso double displacement kinetic mechanism for ChoKα is proposed.•Substrate kinetics and inhibition kinetics of a novel inhibitor were characterised.•Crystal structures suggest that Asp306 stabilises a phospho-enzyme intermediate.•A key feature includes an isomerisation step associated with product release.