Continuous Enzyme-Coupled Assay of Phosphate- or Pyrophosphate-Releasing Enzymes

Abstract

A coupled enzyme assay able to monitor the kinetics of reactions catalyzed by phosphate- or pyrophosphate-releasing enzymes is presented here. The assay is based on the concerted action of inorganic pyrophosphatase (PPase), purine nucleoside phosphorylase (PNPase), and xanthine oxidase (XOD). In the presence of phosphate, PNPase catalyzes the phosphorolysis of inosine, generating hypoxanthine, which is oxidized to uric acid by XOD. The uric acid accordingly formed can be spectrophotometrically monitored at 293 nm, taking advantage of a molar extinction coefficient which is independent of pH between 6 and 9. The coupled assay was tested using DNA polymerases as a model system. The activity of Klenow enzyme was quantitatively determined, and it was found in agreement with the corresponding activity determined by traditional methods. Moreover, the continuous coupled assay was used to determine Km and Vmax of Klenow enzyme, yielding values in good agreement with previous observations. Finally, the coupled assay was also used to determine the activity of partially purified DNA polymerases, revealing its potential use to monitor purification of phosphate- or pyrophosphate-releasing enzymes.

A continuous spectrophotometric enzyme-coupled assay for deoxynucleoside triphosphate triphosphohydrolases

“…To assay the activity of Dgt with AP, we first investigated the activity of AP towards tripolyphosphate, using PNPase and XOD (Scheme 1) to detect the phosphate released according to our previously reported continuous enzyme-coupled assay [23]. However, in agreement with previous observations [25], we observed that AP does catalyze the release of orthophosphate from dGTP (data not shown).…”

“…The hydrolysis of tripolyphosphate by AP was assayed using a previously described enzyme-coupled assay for inorganic phosphate (P i ) [23]. The reactions contained 100 mM Tris-HCl (pH 8), 5 mM MgCl 2 , 0.25 mM inosine, 50 mU/mL Purine nucleoside phosphorylase (PNPase) and 500 mU/mL xanthine oxidase (XOD).…”

“…PPP i and AP concentrations were 100 µM and 50 mU/mL, respectively. The hydrolysis was monitored by quantitating the generated phosphate (P i ) via absorbance changes at 293 nm [23]. …”

“…The protein concentration used was 4 nM; the k cat was calculated at 17.9 ± 0.6 s −1 . The data were fitted to the Hill equation [23]. For the curve of Fig.…”

The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity

“…Oligonucleotides, to be annealed and used as DNA substrates, were synthesized by GenScript. DNA polymerase activity was determined according to our previously described enzyme-coupled assay, which was designed for the continuous spectrophotometric detection of PP i /phosphate [48]. Briefly, reactions were assayed in 100 mM Tris/HCl (pH 8), 5 mM MgCl 2 , 0.25 mM inosine, 100 μM dNTPs and 10, 50 and 500 m-units/ml of inorganic pyrophosphatase (PPase), purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) respectively.…”