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

Pedro, Liliana and Cross, Megan and Hofmann, Andreas and Mak, Tin and Quinn, Ronald J. (2019) Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate kinase. Analytical Biochemistry, 575. pp. 63-69. ISSN 00032697

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 (PvGK), 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 PvGK-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 IC50 curve was generated.

Item Type: Article
Date Deposited: 04 Jun 2019 00:45
Last Modified: 04 Jun 2019 00:45
URI: https://oak.novartis.com/id/eprint/40015

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