Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained 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 yield ADP and TDP. In addition to its vital role in supplying precursors for DNA 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 ATP analog AppNHp, TMP with ADP and the phosphoryl analog AlF(3), TDP and ADP, and the bisubstrate analog TP(5)A were determined. The conformations of the P-loop, the LID region, and the adenine-binding loop vary according 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 to a catalytically unfavorable position, which rotates back in the AlF(3) complex to a position suitable for in-line attack. In the fully closed state observed in the TP(5)A and the TDP-ADP complexes, Asp15 interacts strongly with the 3'-hydroxyl group of TMP.


CONCLUSIONS
The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.
Ostermann
N
N
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Dortmund, 44227, Germany.
Schlichting
I
I
Brundiers
R
R
Konrad
M
M
Reinstein
J
J
Veit
T
T
Goody
R S
RS
Lavie
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000378:metabolism
D000266:Adenylyl Imidodiphosphate; D013938:Thymidine Monophosphate; D000244:Adenosine Diphosphate; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase