A CONTINUOUS VISIBLE SPECTROPHOTOMETRIC ASSAY FOR ASPARTATE-TRANSCARBAMYLASE

A continuous spectrophotometric method for assaying ATCase activity ha s been devised that couples the production of inorganic phosphate from the ATCase-catalyzed reaction to the phosphorolysis reaction catalyze d by purine nucleoside phosphorylase, using a chromophoric nucleotide analogue, methylthioguanosine (MESG). This latter reaction results in a change in extinction coefficient of 11,000 M(-1) cm(-1) at 360 nm, p roviding a means for continuous assay of ATCase activity by spectropho tometry in the visible light region. This Delta epsilon(360) is suffic iently large to allow continuous determination of reaction rates with micromolar levels of carbamyl-phosphate, a feature not offered by othe r currently used assay methods. Other currently available ATCase assay methods typically include fixed-time incubations involving [C-14]Asp that require multiple chromatographic separations, colorimetry requiri ng long incubations with corrosive chemicals in the dark, or relativel y insensitive continuous approaches involving a pH stat or far uv spec trophotometry. This facile, inexpensive MESG-coupled assay can be rout inely applied to studies of ATCase altered by feedback modifiers or by site-specific mutations. Saturation curves for Asp and CP determined by other methods at pH 7 and 8 have been reproduced by the MESG/PNP-co upled approach. The kinetic binding of CP was demonstrated to be nonco operative at low [Asp], i.e., under conditions at which ATCase was pri marily in the T state. Cooperative binding of CP observed under condit ions of saturating [Asp] (i.e., with ATCase in the R state) appears to reflect binding of Asp rather than CP. This arises from a kinetic mec hanism in which CP binds in a strongly preferred manner prior to Asp a nd prior to occurrence of the T-R transition. (C) 1994 Academic Press, Inc.