DB code: S00527

RLCP classification 3.133.90030.394 : Transfer
CATH domain 3.40.50.300 : Rossmann fold Catalytic domain
E.C. 2.7.4.9
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)
3.40.50.300 : Rossmann fold S00547 S00548 S00550 S00554 S00555 S00671 S00672 S00676 S00680 S00682 S00913 S00914 S00301 S00302 S00303 S00304 S00307 S00308 S00305 S00306 S00309 S00310 S00311 M00114 M00199 D00129 D00130 D00540 M00186

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq
P0A720 Thymidylate kinase
TMPK
EC 2.7.4.9
Thymidine monophosphate kinase
dTMP kinase
NP_415616.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_489366.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
O05891 Thymidylate kinase
EC 2.7.4.9
Thymidine monophosphate kinase
dTMP kinase
TMPK
NP_217764.1 (Protein)
NC_000962.3 (DNA/RNA sequence)
NP_337873.1 (Protein)
NC_002755.2 (DNA/RNA sequence)
YP_006516723.1 (Protein)
NC_018143.1 (DNA/RNA sequence)

KEGG enzyme name
dTMP kinase
thymidine monophosphate kinase
thymidylate kinase
thymidylate monophosphate kinase
thymidylic acid kinase
thymidylic kinase
deoxythymidine 5'-monophosphate kinase
TMPK
thymidine 5'-monophosphate kinase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P0A720 KTHY_ECOLI ATP + dTMP = ADP + dTDP. Homodimer.
O05891 KTHY_MYCTU ATP + dTMP = ADP + dTDP. Homodimer. Binds 1 magnesium ion per subunit. This ion is required for catalysis, binding to the active site transiently (at the TMP- binding site), and probably acting as a clamp between the phosphoryl donor and acceptor.

KEGG Pathways
Map code Pathways E.C.
MAP00240 Pyrimidine metabolism

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00002 C00364 C00008 C00363
E.C.
Compound Magnesium ATP dTMP ADP dTDP
Type divalent metal (Ca2+, Mg2+) amine group,nucleotide amide group,nucleotide amine group,nucleotide amide group,nucleotide
ChEBI 18420
15422
17013
16761
18075
PubChem 888
5957
9700
6022
164628
1n5iA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Bound:TMP Unbound Unbound Unbound
1n5jA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Unbound Unbound Bound:TYD Unbound
1n5kA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Bound:TMP Unbound Unbound Unbound
1n5kB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Bound:TMP Unbound Unbound Unbound
1n5lA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Bound:TMP Unbound Unbound Unbound
1n5lB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Unbound Analogue:DPO Bound:TYD Unbound
1g3uA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Bound:TMP Unbound Unbound Unbound
1gsiA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Bound:TMP Unbound Unbound Unbound
1gtvA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Unbound Unbound Bound:TYD Unbound
1gtvB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Bound:TMP Unbound Unbound Unbound
1mrnA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Analogue:T5A(ATP) Analogue:T5A(TMP) Unbound Unbound Unbound
1mrsA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Analogue:5HU Unbound Unbound Unbound
1w2gA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Analogue:THM Unbound Unbound Unbound
1w2gB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Analogue:THM Unbound Unbound Unbound
1w2hA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Analogue:ATM Unbound Unbound Unbound
1w2hB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Analogue:ATM_1209 Analogue:ATM_1210 Unbound Unbound
4tmkA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:T5A(ATP) Analogue:T5A(TMP) Unbound Unbound Unbound
5tmpA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:Z5A(ATP) Analogue:Z5A(ATM) Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
literature [6], [7] & [15]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1n5iA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1n5jA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1n5kA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95; ASP 9;GLU 166(Mg2+ binding) invisible 150-161
1n5kB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1n5lA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95; ASP 9;GLU 166(Mg2+ binding) invisible 150-161
1n5lB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1g3uA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1gsiA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1gtvA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1gtvB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1mrnA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1mrsA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding)
1w2gA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95; ASP 9;GLU 166(Mg2+ binding) invisible 150-161
1w2gB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95;ARG 153 ASP 9;GLU 166(Mg2+ binding) invisible 156-160
1w2hA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95; ASP 9; (Mg2+ binding) invisible 150-167
1w2hB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 13;ARG 95; ASP 9;GLU 166(Mg2+ binding) invisible 153-160
4tmkA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 16;ARG 100;ARG 153 GLU 12(Mg2+ binding)
5tmpA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 16;ARG 100;ARG 153 GLU 12(Mg2+ binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[2]
p.602
[6]
p.3685
[7]
p.14049-14050
[10]
p.638-639, Fig.5 3
[12]
p.97-98
[15]
Fig.6

References
[1]
Resource
Comments
Medline ID
PubMed ID 9253402
Journal Nat Struct Biol
Year 1997
Volume 4
Pages 595-7
Authors Kenyon GL
Title AZT monophosphate knocks thymidylate kinase for a loop.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 9253404
Journal Nat Struct Biol
Year 1997
Volume 4
Pages 601-4
Authors Lavie A, Vetter IR, Konrad M, Goody RS, Reinstein J, Schlichting I
Title Structure of thymidylate kinase reveals the cause behind the limiting step in AZT activation.
Related PDB 1tmk 2tmk
Related UniProtKB P00572
[3]
Resource
Comments
Medline ID
PubMed ID 9256270
Journal Nat Med
Year 1997
Volume 3
Pages 836-7
Authors Hazuda D, Kuo L
Title Failure of AZT: a molecular perspective.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 9256287
Journal Nat Med
Year 1997
Volume 3
Pages 922-4
Authors Lavie A, Schlichting I, Vetter IR, Konrad M, Reinstein J, Goody RS
Title The bottleneck in AZT activation.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 9461164
Journal Nat Med
Year 1998
Volume 4
Pages 132
Authors Balzarini J, Degreve B, De Clercq E
Title Improving AZT efficacy.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 9521686
Journal Biochemistry
Year 1998
Volume 37
Pages 3677-86
Authors Lavie A, Konrad M, Brundiers R, Goody RS, Schlichting I, Reinstein J
Title 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.
Related PDB 3tmk
Related UniProtKB P00572
[7]
Resource
Comments
Medline ID
PubMed ID 9826650
Journal Proc Natl Acad Sci U S A
Year 1998
Volume 95
Pages 14045-50
Authors Lavie A, Ostermann N, Brundiers R, Goody RS, Reinstein J, Konrad M, Schlichting I
Title Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase.
Related PDB 4tmk 5tmp
Related UniProtKB P37345
[8]
Resource
Comments
Medline ID
PubMed ID 10585390
Journal J Biol Chem
Year 1999
Volume 274
Pages 35289-92
Authors Brundiers R, Lavie A, Veit T, Reinstein J, Schlichting I, Ostermann N, Goody RS, Konrad M
Title Modifying human thymidylate kinase to potentiate azidothymidine activation.
Related PDB 1e2q 1e2f 1e2e 1e2g
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID 10666613
Journal Acta Crystallogr D Biol Crystallogr
Year 2000
Volume 56
Pages 226-8
Authors Li de la Sierra I, Munier-Lehmann H, Gilles AM, Barzu O, Delarue M
Title Crystallization and preliminary X-ray analysis of the thymidylate kinase from Mycobacterium tuberculosis.
Related PDB
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID 10873853
Journal Structure Fold Des
Year 2000
Volume 8
Pages 629-42
Authors Ostermann N, Schlichting I, Brundiers R, Konrad M, Reinstein J, Veit T, Goody RS, Lavie A
Title Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 11071809
Journal J Mol Biol
Year 2000
Volume 304
Pages 43-53
Authors Ostermann N, Lavie A, Padiyar S, Brundiers R, Veit T, Reinstein J, Goody RS, Konrad M, Schlichting I
Title 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.
Related PDB 1e99 1e9a 1e9b
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 11469859
Journal J Mol Biol
Year 2001
Volume 311
Pages 87-100
Authors Li de la Sierra I, Munier-Lehmann H, Gilles AM, Barzu O, Delarue M
Title X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution.
Related PDB 1g3u
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID 11914484
Journal Acta Crystallogr D Biol Crystallogr
Year 2002
Volume 58
Pages 607-14
Authors Ursby T, Weik M, Fioravanti E, Delarue M, Goeldner M, Bourgeois D
Title Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals.
Related PDB 1gsi 1gtv
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID 12662932
Journal J Mol Biol
Year 2003
Volume 327
Pages 1077-92
Authors Fioravanti E, Haouz A, Ursby T, Munier-Lehmann H, Delarue M, Bourgeois D
Title Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.
Related PDB 1n5l 1n5k 1n5i 1n5j
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID 12454011
Journal J Biol Chem
Year 2003
Volume 278
Pages 4963-71
Authors Haouz A, Vanheusden V, Munier-Lehmann H, Froeyen M, Herdewijn P, Van Calenbergh S, Delarue M
Title Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism.
Related PDB 1mrn 1mrs
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID 15628853
Journal Biochemistry
Year 2005
Volume 44
Pages 130-7
Authors Fioravanti E, Adam V, Munier-Lehmann H, Bourgeois D
Title The crystal structure of Mycobacterium tuberculosis thymidylate kinase in complex with 3'-azidodeoxythymidine monophosphate suggests a mechanism for competitive inhibition.
Related PDB 1w2g 1w2h
Related UniProtKB

Comments
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.

Created Updated
2002-05-24 2009-02-26