Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine

Kinetic and crystallographic analyses of wild-type Herpes simplex virus typ e 1 thymidine kinase (TKHSV1) and its Y101F-mutant [TKHSV1(Y101F)] acting o n the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been p erformed. The kinetic study reveals a 12-fold K-M increase for thymidine pr ocessed with Y101F as compared to the wild-type TKHSV1 Furthermore, MCT is a substrate for both wild-type and mutant TKHSV1 Its binding affinity for T KHSV1 and TKHSV1(Y101F), expressed as K-i, is 11 mu M and 51 mu M, respecti vely, whereas the K-i for human cytosolic thymidine kinase is as high as 1. 6 mM, rendering TKHSV1 a selectivity filter for antiviral activity. Moreove r, TKHSV1(Y101F) shows a decrease in the quotient of the catalytic efficien cy (k(cat)/K-M) of dT over MCT corresponding to an increased specificity fo r MCT when compared to the wild-type enzyme. Crystal structures of wild-typ e and mutant TKHSV1 in complex with MCT have been determined to resolutions of 1.7 and 2.4 Angstrom, respectively. The thymine moiety of MCT binds Lik e the base of dT while the conformationally restricted bicyclo[3.1.0]-hexan e, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond p attern around the sugar-like moiety differs from that of thymidine, reveali ng the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance -repellent drugs for antiviral therapy, and mutant Y101F, in combination wi th MCT, opens new possibilities for gene therapy.