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J Biol Chem, Vol. 274, Issue 45, 31967-31973, November 5, 1999

Substrate Diversity of Herpes Simplex Virus Thymidine Kinase
IMPACT OF THE KINEMATICS OF THE ENZYME

Beatrice D. Pilger, Remo Perozzo, Frank Alber^§¶, Christine Wurth, Gerd Folkers, and Leonardo Scapozza

From the  Department of Pharmacy, Swiss Federal Institute of Technology, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland and the ^§ Instituto Nazionale per la Fisica della Materia and ^¶ International School for Advanced Studies, Via Beirut 4, 34014 Trieste, Italy

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches.

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