Biophysical characterization of vaccinia virus thymidine kinase substrate utilization.
To provide information for the development of new antiviral compounds that inhibit orthopoxviruses, further characterization of the kinetics and thermodynamics that underlie substrate utilization reactions of vaccinia virus thymidine kinase (VVTK) has been undertaken. The kinetics of 2'deoxythymidine phosphorylation by VVTK and the thermodynamics of complex formation between VVTK and the substrate 2' deoxythymidine were determined using spectroscopic and calorimetric techniques. These studies demonstrated that kinetic parameters for 2' deoxythymidine phosphorylation by VVTK were 25 microM and 0.2s(-1) for K(m) and k(cat), respectively. The enthalpy change, Delta H, for the enzyme catalyzed reaction is -18.1 kcal/mol. Thermodynamic studies for the formation of the enzyme substrate complex demonstrated a binding affinity (K(a)) of 4 x 10(4)M(-1), an enthalpy change for binding (Delta H) of -17.4 kcal/mol, and a reaction stoichiometry of two molecules of substrate binding to each enzyme tetramer. Kinetic and thermodynamic data were in agreement (K(a) approximately 1/K(m)) and showed similarities to literature values reported for herpes simplex virus thymidine kinase (HSV-TK) and human thymidine kinase 1 (hTK1) with respect to k(cat) but not with respect to K(m). The K(m) value found for VVTK in this study is nearly two orders of magnitude larger than the values reported for the hTK1 and the HSV TK enzymes.
Smith
Robert F
RF
Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ 86011-5698, USA.
Freyer
Matthew W
MW
Lewis
Edwin A
EA
D002151:Calorimetry; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013816:Thermodynamics; D013936:Thymidine / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D013937:Thymidine Kinase; C092717:thymidine kinase 1; D013936:Thymidine