Enzyme cycling method using hypoxanthine-guanine phosphoribosyltransferase: a highly sensitive assay for pyrophosphate†

Abstract

We developed a novel enzyme cycling method using hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) from Hungateiclostridium thermocellum. This method exploits the reversible nature of the HGPRT reaction, catalyzing both forward and reverse reactions in the presence of excess inosine 5′-monophosphate (IMP) and guanine (Gua), similar to the established purine nucleoside phosphorylase (PNP)-based method. Real-time detection was achieved by coupling the reaction with commercially available xanthine dehydrogenase (XDH) (EC 1.17.1.4) in the presence of nicotinamide adenine dinucleotide (NAD⁺). The reaction exhibited high efficiency, with a cycling rate constant of approximately 60 min⁻¹ or higher at an HGPRT concentration of 1 U mL⁻¹. Additionally, we demonstrated a preliminary application of this XDH-coupled enzyme cycling reaction for the determination of pyrophosphate (PPi).