A spectrophotometric assay for quantitative determination of kcat of herpes simplex virus type 1 thymidine kinase substrates.
A simple method to determine the in vitro catalytic turnover constant of several substrates of herpes simplex virus type 1 thymidine kinase is presented 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 thymidine kinase activity in the presence of unlabeled substrates. A clear correlation between the catalytic turnover constant and the rate of decrease in absorbance over time during the assay has been demonstrated. Exploiting this correlation, this method has been used to determine rapidly and precisely the catalytic turnover constant of antiviral lead compounds not readily available in the radioactive labeled form.
Schelling
P
P
Department of Applied BioSciences, Swiss Federal Institute of Technology (ETH), Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
Folkers
G
G
Scapozza
L
L
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D002384:Catalysis; D015195:Drug Design; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000378:metabolism; D011817:Rabbits; D011874:Radiometry; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013937:Thymidine Kinase / Q000378:metabolism*
D000998:Antiviral Agents; D009243:NAD; D010728:Phosphoenolpyruvate; D007770:L-Lactate Dehydrogenase; D013937:Thymidine Kinase; D011770:Pyruvate Kinase