Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase

Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14045-50. doi: 10.1073/pnas.95.24.14045.


The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P1-(5'-adenosyl)-P5-(5'-thymidyl)pentaphosphate and P1-(5'-adenosyl)P5-[5'-(3'-azido-3'-deoxythymidine)] pentaphosphate have been solved to 2.0-A and 2.2-A 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 (kcat) in comparison to the physiological substrate dTMP, reduction of kcat 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antiviral Agents / metabolism*
  • Catalytic Domain
  • Crystallography, X-Ray
  • Dideoxynucleotides
  • Escherichia coli / enzymology*
  • Humans
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleoside-Phosphate Kinase / chemistry*
  • Nucleoside-Phosphate Kinase / metabolism*
  • Phosphorylation
  • Protein Structure, Secondary*
  • Saccharomyces cerevisiae / enzymology
  • Sequence Alignment
  • Zidovudine / analogs & derivatives*
  • Zidovudine / metabolism


  • Antiviral Agents
  • Dideoxynucleotides
  • 3'-azido-2',3'-dideoxyuridine 5'-monophosphate
  • Zidovudine
  • Nucleoside-Phosphate Kinase
  • dTMP kinase

Associated data

  • PDB/4TMK
  • PDB/5TMP