A spectro photometric assay for quantitative determination of k(cat) of herpes simplex virus type 1 thymidine kinase substrates

A simple method to determine the in vitro catalytic turnover constant of se veral substrates of herpes simplex virus type I thymidine kinase is present ed in this study. The method is based on a continuous spectroscopic enzyme- coupled assay and allows one to monitor the herpes simplex virus type 1 thy midine kinase activity in the presence of unlabeled substrates. A clear cor relation between the catalytic turnover constant and the rate of decrease i n absorbance over time during the assay has been demonstrated. Exploiting t his correlation, this method has been used to determine rapidly and precise ly the catalytic turnover constant of antiviral lead compounds not readily available in the radioactive labeled form. (C) 2001 Academic Press.