1E2L

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

Summary for 1E2L

• Entry DOI: 10.2210/pdb1e2l/pdb
• Related: 1E2H 1E2I 1E2J 1E2K 1E2M 1E2N 1E2P 1KI2 1KI4 1KI5 1KI6 1KI7 1KI8 1KIM 1VTK 2VTK 3VTK
• Descriptor: THYMIDINE KINASE, SULFATE ION, 1-[4-HYDROXY-5-(HYDROXYMETHYL)BICYCLO[3.1.0]HEX-2-YL]-5-METHYLPYRIMIDINE-2,4(1H,3H)-DIONE, ... (4 entities in total)
• Functional Keywords: thymidine kinase, antiviral drug, enzyme-prodrug gene therapy, sugar ring pucker
• Biological source: HERPES SIMPLEX VIRUS (TYPE 1 / STRAIN 17)
• Total number of polymer chains: 2
• Total formula weight: 72222.83

Authors

Vogt, J.,Scapozza, L.,Schulz, G.E. (deposition date: 2000-05-23, release date: 2000-08-18, Last modification date: 2023-12-06)

Primary citation

Prota, A.,Vogt, J.,Pilger, B.,Perozzo, R.,Wurth, C.,Marquez, V.,Russ, P.,Schulz, G.E.,Folkers, G.,Scapozza, L.
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
Biochemistry, 39:9597-, 2000

PubMed Abstract: 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.

PubMed: 10924157
DOI: 10.1021/BI000668Q

Experimental method

X-RAY DIFFRACTION (2.4 Å)