Nucleoside analogue prodrugs are dependent on efficient intracellular stepwise phosphorylation to their triphosphate form to become therapeutically active. In many cases it is this activation pathway that largely determines the efficacy of the drug. To gain further understanding of the determinants for efficient conversion by the enzyme thymidylate kinase (TMPK) of clinically important thymidine monophosphate analogues to the corresponding diphosphates, we solved the crystal structures of the enzyme, with either ADP or the ATP analogue AppNHp at the phosphoryl donor site, in complex with TMP, AZTMP (previous work), NH2TMP, d4TMP, ddTMP, and FLTMP (this work) at the phosphoryl acceptor site. In conjunction with steady-state kinetic data, our structures shed light on the effect of 3'-substitutions in the nucleoside monophosphate (NMP) sugar moiety on the catalytic rate. We observe a direct correlation between the rate of phosphorylation of an NMP and its ability to induce a closing of the enzyme's phosphate-binding loop (P-loop). Our results show the drastic effects that slight modifications of the substrates exert on the enzyme's conformation and, hence, activity and suggest the type of substitutions that are compatible with efficient phosphorylation by TMPK.

The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme. The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation.

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 AIP analog AppNHp, TMP with ADP and the phosphoryl analog AIF(3), TDP and ADP, and the bisubstrate analog TP,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 AIF3 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.

Based on the knowledge of the crystal structures of yeast and Escherichia coli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficiently than either the yeast or the highly homologous human enzyme, we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties. These modified enzymes have properties that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency virus (HIV) replication. In particular, insertion of the lid domain of the bacterial TmpK into the human enzyme results in a pronounced change of the acceptance of AZT-MP such that it is now phosphorylated even faster than TMP.

Convenient syntheses of new fluorescent derivatives of guanosine nucleotides are described. The analogs bear a fluorescent group attached by an aliphatic linker containing 2 or 4 methylene groups on the N-2 position of the guanine base. They interact with the small GTPases H-Ras and cdc42 with an accompanying increase in fluorescence yield and display kinetic constants which are similar to those of unmodified nucleotides. The derivatives thus appear to be attractive alternatives to the widely used 2'(3')-O-methylanthraniloyl derivatives and are likely to be useful for GTPases which do not tolerate modifications of the sugar moiety of the nucleoside.

The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P 1 -(5′-adenosyl)- P 5 -(5′-thymidyl)pentaphosphate and P 1 -(5′-adenosyl)P 5 -[5′-(3′-azido-3′-deoxythymidine)] pentaphosphate have been solved to 2.0-Å and 2.2-Å resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3′-azido-3′-deoxythymidine 5′-monophosphate (AZT-MP) at a 200-fold reduced turnover number ( k cat ) in comparison to the physiological substrate dTMP, reduction of k cat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3′-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3′-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3′ substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli ) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.