Wild K, Bohner T, Folkers G, Schulz G E
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg im Breisgau, Germany.
Protein Sci. 1997 Oct;6(10):2097-106. doi: 10.1002/pro.5560061005.
Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments.
1型单纯疱疹病毒胸苷激酶（TK）以N端截短但具有完全活性的形式结晶。TK与ATP位点的ADP以及底物位点的脱氧胸苷-5'-单磷酸（dTMP）、脱氧胸苷（dT）或碘苷-5'-磷酸（5-碘-dUMP）形成的复合物结构分别精修至2.75 Å、2.8 Å和3.0 Å分辨率。TK催化dT磷酸化生成酯，催化dTMP磷酸化生成酸酐。所呈现的TK结构表明这两种作用模式之间只有微小差异。Glu83在酯反应中作为通用碱。Arg163在酯形成过程中停靠在内部天冬氨酸处，在酸酐形成过程中结合dTMP的α-磷酸。结合的脱氧胸苷在碱基的5位留下一个35 ų的空腔，在2位留下两个隔离水分子。空腔和水分子降低了底物特异性，以至于TK能够磷酸化各种在药物应用中有用的底物类似物。TK在结构上与著名的核苷单磷酸激酶同源，但含有大量额外的肽段。
