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.
The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme. The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation.
Ostermann
N
N
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Str. 11 D-44227 Dortmund, Germany.
Lavie
A
A
Padiyar
S
S
Brundiers
R
R
Veit
T
T
Reinstein
J
J
Goody
R S
RS
Konrad
M
M
Schlichting
I
I
D019380:Anti-HIV Agents / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D054306:Dideoxynucleotides; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010766:Phosphorylation; D011355:Prodrugs / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism*
D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D009711:Nucleotides; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase