TMPK

Related Reading

TMPK (EC 2.7.4.9, thymidine monophosphate kinase) belongs to the the class of transferases, is a phosphate group transferase. The systematic name of this enzyme is ATP:dTMP phosphotransferase. Other names in common use include thymidine monophosphate kinase, thymidylate kinase, thymidylate monophosphate kinase, thymidylic acid kinase thymidylic kinase, deoxythymidine 5'-monophosphate kinase, TMPK, and thymidine 5'-monophosphate kinase. TMPK catalyzes the phosphorylation of thymidine 5’-monophosphate (dTMP) to produce thymidine 5’-diphosphate (dTDP) in the presence of ATP and Mg²⁺. dTDP is further phosphorylated by nucleoside-diphosphate kinase (NDK) to produce thymidine 5’-triphosphate (dTTP). As a substrate for TMPK, dTMP either comes from salvage pathway or from de novo pathway, so TMPK acts on the junction of de novo and salvage pathways of dTTP synthesis.

Structure

TMPK belongs to the NMP kinase family, which has a highly conserved fold but has fewer conserved sequences. TMPKs are all homodimers, each subunit consisting of 7-11 α-helices surrounding a central 5 parallel β-strands. TMPK contains three domains: the ligand-induced degradation (LID) domain, the NMP binding site and the CORE domain. The LID domain is flexible, partially surrounding the phosphate donor, allowing the transfer of phosphate groups. The CORE domain comprises a phosphate-binding loop (P-loop) and an ATP binding site. P-loop includes the structural elements required for substrate recognition and catalysis, and the P-loop binds and localizes to α and β phosphoryl groups of ATP. All known TMPKs have a similar CORE domain.

Figure 1. Structure of TMPK. (Cui Q. 2013)

TMPK is classified into two types based on the position of the basic residue of its active site. Type I TMPK is mainly derived from eukaryotes, and they have basic residues in addition to the invariant lysine accident in P-loop. Type II TMPK is mainly derived from prokaryotes, which lack basic residues in the P-loop, but have basic residues with similar effects in the LID domain. It is worth neither noting that M. tuberculosis TMPK is neither of type I nor type II, and its catalytic mechanism is also different.

Catalytic Mechanism

TMPK is a nucleoside monophosphate kinase that catalyzes the conversion of dTMP to dTDP in the presence of ATP and Mg²⁺: ATP + thymidine 5'-phosphate ⇌ ADP + thymidine 5'-diphosphate. As a substrate for TMPK, dTMP is mainly derived from salvage pathways and de novo synthesis pathways, so TMPK is functionally localized at the junction of salvage pathways and de novo synthesis pathways. The thymidine base of dTMP binds between the Phe72 and a face made by helix 5 of the P-loop. The studies of NMP family found that substrates induce changes in the conformation of the NMP binding site, and several conformational states of TMPK associated with the catalytic mechanism of the enzyme were discovered. In the absence of a ligand, TMPK is open state, in which the LID domain is disordered. In the presence of dTMP, TMPK is partially closed and the LID domain is disordered in most structures. In the case where both substrates coexist, TMPK is a fully closed state and the LID domain is ordered. During the catalytic process, P-loop movement causes the conformation change from an open state to a closed state, bringing the nucleoside triphosphate moiety close to the phosphoryl acceptor.

Figure 2. Thymidylate kinase functional pathway. (Lavie A. 2004)

Figure 3. Mechanism of dTMP bound to TMPK. (Lavie A. 2004)

Application

As a key enzyme in dTTP synthesis, TMPK can be a potential drug target in a variety of diseases, especially in infectious diseases. Interrupting dTTP metabolism may be used to prevent the development of such diseases. When the patient took the anti-HIV nucleoside analog AZT, it was first phosphorylated by thymidine kinase to 3'-azido-3'-deoxythymidine monophosphate (AZTMP). AZTMP is further phosphorylated by TMPK into 3'-azido-3'-deoxythymidine diphosphate (AZTDP). AZTMP is a poor substrate for human TMPK (hTMPK), so conversion to AZTDP is the rate-limiting step in the process of activating. The final activation step is carried out by base non-specific nucleoside diphosphate kinase (NDK) to form 3'-azido-3'-deoxythymidine triphosphate (AZTTP). AZTTP has the 3' azido group replacing the 3'-OH of ribose, which terminates DNA synthesis or acts as an inhibitor of DNA polymerase to block DNA synthesis. hTMPK is an important enzyme controlling the rate of the process throughout the activation process.

TMPK inhibition is an effective strategy for the discovery of infectious disease drugs, such as bacterial and parasitic infections. Subtle differences in hTMPK and pathogen TMPK active sites provide an opportunity to design selective inhibitors. In addition, hTMPK inhibitors can be used in combination with anti-cancer agents to treat some complex cancer diseases.

References

1. Cui, Q., Shin, W.S., Luo, Y., Tian, J., Cui, H., Yin, D. Thymidylate kinase: an old topic brings new perspectives. Curr Med Chem, 2013, 20(10):1286-305.
2. Lavie, A., Konrad, M. Structural requirements for efficient phosphorylation of nucleotide analogs by human thymidylate kinase. Mini Rev Med Chem, 2004, 4(4):351-9.