Insights into the phosphoryltransfer mechanism of human thymidylate kinasegained from crystal structures of enzyme complexes along the reaction coordinate

Background: Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryltransfer between ATP and TMP to yie ld ADP and TDP. In addition to its vital role in supplying precursors for D NA synthesis, human TMPK has an important medical role participating in the activation of a number of anti-HIV prodrugs. Results: Crystal structures of human TMPK in complex with TMP and ADP, TMP and the AIP analog AppNHp, TMP with ADP and the phosphoryl analog AIF(3), T DP and ADP, and the bisubstrate analog TP,A were determined. The conformati ons of the P-loop, the LID region, and the adenine-binding loop vary accord ing to the nature of the complex. Substitution of ADP by AppNHp results in partial closure of the P-loop and the rotation of the TMP phosphate group t o a catalytically unfavorable position, which rotates back in the AIF3 comp lex to a position suitable for in-line attack. In the fully closed state ob served in the TP(5)A and the TDP-ADP complexes, Asp15 interacts strongly wi th the 3'-hydroxyl group of TMP. Conclusions: The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermedia tes occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human T MPK appears to be determined by structural changes required to achieve cata lytic competence and it is suggested that a mechanism might exist to accele rate the activity.