Mutagenesis of non-conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2

• Type of Material: Article
• Authors: Monica L. Gerth, Emory University; Stefan Lutz, Emory University
• Language: English
• Date: 2007-03-16
• Publisher: Elsevier: 12 months
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2007 Elsevier Inc. All rights reserved.
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1016/j.bbrc.2007.01.070
• Title of Journal or Parent Work: Biochemical and Biophysical Research Communications
• ISSN: 0006-291X
• Volume: 354
• Issue: 3
• Start Page: 802
• End Page: 807
• Grant/Funding Information: We acknowledge financial support in part by the National Institutes of Health (GM69958), as well as by a grant to the Emory Center for AIDS Research (AI050409) from the National Institutes of Health and by institutional funding from the Emory University Health Science Center.
• Keywords: Human thymidine kinase 2; site-directed mutagenesis; deoxyribonucleoside kinase; nucleoside analog
• Research Categories: Chemistry, General

Abstract

Human thymidine kinase 2 (TK2) is critical for the nucleotide salvage pathway and phosphorylation of nucleoside analog prodrugs in vivo; however, it remains poorly studied because of difficulties expressing it heterologously. TK2 is strictly pyrimidine-specific, whereas its phylogenetic relative, the Drosophila melanogaster deoxyribonucleoside kinase (DmdNK), shows higher activity and broader specificity towards both pyrimidines and purines. These differences are counterintuitive, as only two of 29 active site residues differ in the two enzymes: F80 and M118 in DmdNK are L78 and L116 in TK2. In addition to reporting an optimized protocol for the expression and purification of TK2, we have used site-directed mutagenesis to introduce the DmdNK-like amino acids into TK2, and characterized the three resulting enzymes (L78F-TK2, L116M-TK2 and L78F/L116M-TK2). These mutations improve the KM for thymidine, increasing the catalytic activity of L78F/L116M-TK2 4.4-fold, yet leave activity for deoxycytidine or the purine nucleosides unchanged.