Abstract

A new continuous coupled UV-spectrophotometric assay is described for two phosphate releasing enzymes, aspartate transcarbamylase and ATPase of Herpes Simplex Virus. Phosphate release is coupled to the phosphorolysis of the nucleoside analogue 7-methylinosine catalyzed by purine nucleoside phosphorylase. When this reaction is monitored at 291 nm, the coupled assay can readily detect 10 nmol P$\rm\sb{i}$ released/min. Our method offers advantages over a recently reported continuous assay devised for measuring ATCase activity using the nucleoside analogue methylthioguanosine as the linking substrate. In contrast to MESG, m$\sp7$Ino is easily and inexpensively synthesized and is also commercially available. The spectrophotometric signal at 291 nm, produced by the difference in the extinction coefficients between nucleoside substrate and the base product, is significant over a much wider pH range than the signal difference between MESG and its phosphorolysis product at 360 nm. Saturation curves for aspartate and carbamyl phosphate and pH rate profiles have been reproduced using the PNPase/m$\sp7$Ino coupled assay. Initial velocity patterns constructed over micromolar to millimolar concentrations of aspartate and CbmP yield four kinetic parameters simultaneously. To further illustrate the application of this coupled assay, kinetic parameters were determined for the DNA-dependent ATPase reaction of HSV helicase-primase.