Fig. 1.

Continuous P_i-sensor Nudix hydrolase assay. (A) Nudix hydrolase, coupling enzyme (phosphatase), and P_i-sensor are incubated together with the putative Nudix substrate. Activity is monitored by increase in fluorescence. The choice of coupling enzyme depends on the X moiety (which could be hydrogen, phosphate, or other moieties; see below) of the substrate. The coupling enzyme can be either inorganic pyrophosphatase (PPase) or alkaline phosphatase (APase). (B) Two substrate classes are shown. (1) When X is a hydrogen (i.e., nucleotide diphosphate), a monophosphate (i.e., nucleotide triphosphate), or a polyphosphate (e.g., nucleotide tetraphosphate), Nudix enzyme-catalyzed hydrolysis yields either free phosphate or polyphosphate (e.g., pyrophosphate as shown) as one of the products. In the latter case, pyrophosphatase effects the release of free phosphate. (2) For other X moieties, hydrolysis will not yield free phosphate but will yield at least one phosphate ester as a product. Here, APase catalyzes the hydrolysis to yield free phosphate.