1e2k

From Proteopedia

Revision as of 13:58, 5 November 2007

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

Overview

Kinetic and crystallographic analyses of wild-type Herpes simplex virus, type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)], acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT), have been performed. The kinetic study reveals a 12-fold K(M) increase for, thymidine processed with Y101F as compared to the wild-type TK(HSV1)., Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1)., Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human, cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a, selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows, a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT, over MCT corresponding to an increased specificity for MCT when compared, to the wild-type enzyme. Crystal structures of wild-type and mutant, TK(HSV1) in complex with MCT have been determined to resolutions of 1.7, and 2.4 A, respectively. The thymine moiety of MCT binds like the base of, dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking, the sugar moiety, assumes a 2'-exo envelope conformation that is flatter, than the one observed for the free compound. The hydrogen bond pattern, around the sugar-like moiety differs from that of thymidine, revealing 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 with MCT, opens new possibilities for gene therapy.

About this Structure

1E2K is a Single protein structure of sequence from Human herpesvirus 4 with SO4 and TMC as ligands. Active as Thymidine kinase, with EC number 2.7.1.21 Structure known Active Sites: AC1 and AC2. Full crystallographic information is available from OCA.

Reference

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., Prota A, Vogt J, Pilger B, Perozzo R, Wurth C, Marquez VE, Russ P, Schulz GE, Folkers G, Scapozza L, Biochemistry. 2000 Aug 8;39(31):9597-603. PMID:10924157

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