A continuous visible spectrophotometric assay for aspartate transcarbamylase

A continuous spectrophotometric method for assaying ATCase activity has been devised that couples the production of inorganic phosphate from the ATCase-catalyzed reaction to the phosphorolysis reaction catalyzed 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, providing a means for continuous assay of ATCase activity by spectrophotometry in the visible light region. This delta epsilon 360 is sufficiently large to allow continuous determination of reaction rates with micromolar levels of carbamyl-phosphate, a feature not offered by other currently used assay methods. Other currently available ATCase assay methods typically include fixed-time incubations involving [14C]Asp that require multiple chromatographic separations, colorimetry requiring long incubations with corrosive chemicals in the dark, or relatively insensitive continuous approaches involving a pH stat or far uv spectrophotometry. This facile, inexpensive MESG-coupled assay can be routinely 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-coupled approach. The kinetic binding of CP was demonstrated to be non-cooperative at low [Asp], i.e., under conditions at which ATCase was primarily in the T state. Cooperative binding of CP observed under conditions of saturating [Asp] (i.e., with ATCase in the R state) appears to reflect binding of Asp rather than CP.(ABSTRACT TRUNCATED AT 250 WORDS)