Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate kinase.

Abstract The development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (Pv GK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the Pv GK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z' factor of 0.6. For the best inhibitor, an IC 50 curve was generated. Graphical abstract Image 1 Highlights • Simple HPLC separation of nucleotides involved in the guanylate kinase reaction. • Direct and unambiguous determination of percent conversion of substrate into product. • Successful application to Plasmodium vivax guanylate kinase (Pv GK) activity studies. • Reaction catalyzed by Pv GK found to be associated with atypical kinetics. • Robust and reproducible inhibition assay for compound screening. [ABSTRACT FROM AUTHOR]