Kinetic studies of herpes simplex virus type 1-encoded thymidine and thymidylate kinase, a multifunctional enzyme.

Kinetic studies of the reactions catalyzed by herpes simplex virus type l-encoded thymidine and thymidylate multifunctional kinase indicate that the addition of substrates (thymidine and ATP*Mg’+, or thymidylate and ATP l Mg2+) to the enzyme is random, while release of the products (thymidylate and ADP*M8+, or thymidine diphosphate and ADP*Mg+) is ordered, with ADP.Mg2+ being released first. The dissociation constants of ADP*M&+ from the enzyme l ADP. Mg2+ binary complex and the enzyme l thymidine l ADP l Mg2’ ternary complex were found to be 28 PM, and 2.2 PM, respectively. At saturating thymidine concentrations (loo-fold K,), thymidylate and thymidine diphosphate activate the phosphorylation of thymidine, but at low thymidine concentrations (below 3or 4-fold K,) its phosphorylation is inhibited. A separate binding site for thymidylate and thymidine diphosphate which regulates the phosphorylation of thymidine is suggested. The regulatory binding site for thymidylate has a K, of 47 pM, and is present even when more than 70% of enzyme activity is destroyed by heat denaturation. The K, for thymidine diphosphate binding to the regulatory site is 100 CM. A direct transfer of the pand y-phosphate from ATP*Mg2+ to thymidine to form thymidine diphosphate was negated, and direct channeling of dThd to dTDP appears to be minimal. Inhibition studies indicated that both thymidine kinase and thymidylate kinase activities either have the same active site or part of two active sites overlap. The second step of the overall reaction (ATP=Mg2+ + thymidylate + ADP.M$+ + thymidine diphosphate) was characterized by a higher K,,, and lower V,, for both substrates than for the first part of the reaction (ATP.Mg’+ + thymidine -+ ADP. Mg2+ + thymidylate). This may be due to the difference in steric volume of thymidylate relative to thymidine at the active site. Hill coefficients for thymidine, thymidylate, ATP. M8’ (thymidine), and ATP*M8+ (thymidylate) were determined to be 1.07, 0.87, 0.90, and 1.07, respectively. No cooperative binding was observed.