Coupled spectrofluorometric assay for aminoglycoside phosphotransferases

Abstract

In order to make accurate kinetic measurements for the substrates of aminoglycoside (AG) phosphotransferases (APHs), we have developed an assay which overcomes many of the limitations of currently used assays. We have adapted the coupled spectrophotometric assay ( P. R. Goldman and D. B. Northrop (1976) Biochem. Biophys. Res. Commun. 68, 230–236) for use in a spectrofluorometer. At an excitation wavelength of 340 nm, NADH will emit an intensity peak at 450 nm; NAD does not emit under these conditions. Our assay can accurately measure differences of 0.25 μ m. For the APH(3′)-II encoded on Tn5, we have redetermined the K m 's for the AGs, amikacin (AK), kanamycin (KM), and ribostamycin (Rib), and for ATP. Our values for AK (76 μ m) were lower than those derived from the spectrophotometric assay; for KM and Rib we obtained K m values of 5.1 and 3.6 μ m, respectively. These values were well below the limit of accuracy (10 μ m) for the spectrophotometric assay. In addition, we have begun characterization of an APH from a clinical isolate with a low K m for AK. Thus far, we have determined that this enzyme has K m 's of approximately 1 μ m for both AK and KM. These results show that the assay is well suited for accurate determinations of kinetic constants for low- K m substrates of APH enzymes.