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.

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.Phosphorylation of AZT-MP by thymidylate kinase (TmpK; EC 2.7.4.9, ATP:dTMP phosphotransferase) has been implicated as the rate-limiting step in the activation pathway of the anti-HIV prodrug 3Ј-azido-3Ј-deoxythymidine (AZT). AZT must be phosphorylated by cellular enzymes to azidothymidine triphosphate before it can exert its antiviral effect by inhibiting HIV reverse transcriptase and acting as a chain terminator of nascent DNA strands. The bottleneck of AZT activation lies in the second phosphorylation step, the step that adds a phosphate group to azidothymidine monophosphate (AZT-MP) to yield azidothymidine diphosphate and is catalyzed by TmpK. Cells treated with AZT accumulate the toxic AZT-MP (1-3) in millimolar concentration (4, 5), and thus the active AZT triphosphorylated metabolite is at very low concentration. This situation is only slightly improved upon coinfection with herpes simplex virus I thymidine kinase-carrying vectors that efficiently phosphorylate the antiviral drugs acyclovir or ganciclovir but not AZT or AZT-MP (6, 7).Therefore, we set out to understand the structural basis for the slow activation of AZT-MP. The structures of the yeast TmpK (TmpK yeast ) complexed with either the physiological substrate d...

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.

The crystal structure of yeast thymidylate kinase (TmpK) complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) was determined at 2.0 A resolution. In this complex, TmpK adopts a closed conformation with a region (LID) of the protein closing upon the substrate and forming a helix. The interactions of TmpK and TP5A strongly suggest that arginine 15, which is located in the phosphate binding loop (P-loop) sequence, plays a catalytic role by interacting with an oxygen atom of the transferred phosphoryl group. Unlike other nucleoside monophosphate kinases where basic residues from the LID region participate in stabilizing the transition state, TmpK lacks such residues in the LID region. We attribute this function to Arg 15 of the P-loop. TmpK plays an important role in the phosphorylation of the AIDS prodrug AZT. The structures of TmpK with dTMP and with AZT-MP [Lavie, A., et al. (1997) Nat. Struct. Biol. 4, 601-604] implicate the movement of Arg15 in response to AZT-MP binding as an important factor in the 200-fold reduced catalytic rate with AZT-MP. TmpK from Escherichia coli lacks this arginine in its P-loop while having basic residues in the LID region. This suggested that, if such a P-loop movement were to occur in the E. coli TmpK upon AZT-MP binding, it should not have such a detrimental effect on catalysis. This hypothesis was tested, and as postulated, E. coli TmpK phosphorylates AZT-MP only 2.5 times slower than dTMP.

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