AZT monophosphate knocks thymidylate kinase for a loop.
Structure of thymidylate kinase reveals the cause behind the limiting step in AZT activation.
Failure of AZT: a molecular perspective.
Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) at 2.0 A resolution: implications for catalysis and AZT activation.
Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase.
Modifying human thymidylate kinase to potentiate azidothymidine activation.
Crystallization and preliminary X-ray analysis of the thymidylate kinase from Mycobacterium tuberculosis.
Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.
Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP.
X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution.
Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals.
Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.
Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism.
This enzyme, thymidylate kinase (TmpK), was very important in terms of the medicine for AIDS, azidothymidine (AZT) [1],[2],[3]. AZT is a prodrug that must be converted by cellular enzymes, such as thymidine kinase and TmpK, to the active triphosphate form [3]. According to the paper [12], the catalytic residues and magnesium binding site of the enzyme are so various and different between those from different organisms (bacteria, archaebacteria, eukaryote, and even mammal). The paper [7] mentioned that there are two types of TmpKs. Type I TmpKs stabilize the negative charge in the transition state by having the positively charged guanidinium group originating from the P-loop (yeast enzyme), whilst in the type II enzymes the guanidinium group originates from the LID region (E. coli enzyme). Usually, NMP kinases have catallytically essential residues in the LID region. Furthermore, TmpKs use only a few basic residues to interact with the transferred phosphoryl group, whereas other NMP kinases, such as adenylate kinase or uridylate kinase, use five [7]. For example, yeast TmpK appears to have the catalytic residues in the P-loop (Arg15, Lys19), whilst it lacks basic residues in its LID region [2], [6]. Moreover, the conserved arginine residue, Arg94 in yeast TmpK (Arg100 in E. coli enzyme) also appears to interact with both the gamma-phosphate of ATP and the phosphate of the nucleoside monophosphate (i.e., the transferred phosphate and the acceptor phosphate) [7]. In contrast, in the E. coli TmpK, Arg153 from the LID region and the conserved arginine, Arg100, as well as the lysine residue in the P-loop are involved in catalysis [7]. However, in the case of human TmpK, the arginine from the P-loop does not interact with the transferred phosphoryl group. Only the conserved arginine, Arg97, plays a role in catalysis, as well as the lysine from the P-loop, and acts as a clamp to bring the donor and acceptor [10]. The archaebacterial enzyme displayed very different features from the other TmpK enzymes [12], [14]. Whereas magnesium binding site for the human TmpK was located along the ATP-binding site, between the beta and gamma phosphate groups, the archaebacterial enzyme indicated the magnesium ion was positioned in the TMP-binding site [12], [14]. In addition, this ion plays an essential role in catalysis [14]. Firstly, the cation provides a strong electrostatic potential to attract the gamma-phosphate group of ATP sufficiently close to the alpha-phosphate group of TMP for phosphoryl transfer. Secondly, the crystal structures suggested that binding of ATP involves a direct coordination of the gamma-phosphate group of ATP onto the metal, which, locked tightly in the active site, is able to play the role of a clamp between the phosphoryl donor and acceptor [14]. As for the catalytic residues in the archaebacterial enzyme, Arg95 seems to neutralise the electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions, in concert with the magnesium ion [14]. Thus, catalysis by arginine can occur as long as the interaction with the transferred phosphate group is possible, regardless of where the arginine is located in the secondary structure [7]. The paper [7] also identified four different situations for catalytic arginines in phosphate-transferring enzymes; the catalytic arginine can be located (1) in the P-loop, (2) in other region (such as LID region), (3) in a different domain of a multidomain protein (see heterotrimeric G proteins), (4) in other protein capable of interacting with the phosphate-transferring protein (see Ras-RasGAP). This can explain why TmpK enzymes have only a few basic residues for the catalysis, whereas the other homologous NMP kinases have as many as five [7]. Furthermore, the paper [15] mentioned that the role of conserved serine residue (Ser99 in bacterial enzyme; PDB code, 1n5l, etc.) seems to be protonating the transferred PO3 group through Arg95 and Asp9. However, considering the structure with ligand, it seems unlikely.