Tight Binding of Deoxyribonucleotide Triphosphates to Human Thymidine Kinase 2 Expressed in Escherichia coli. Purification and Partial Characterization of Its Dimeric and Tetrameric Forms

• Published in: Biochemistry (Easton) 2003-12, Vol.42 (51), p.15158-15169
• Main Authors: Barroso, João Filipe, Elholm, Morten, Flatmark, Torgeir
• Format: Article
• Language: English
• Subjects: Binding Sites; Cell Line, Tumor; Chaperonin 10 - biosynthesis; Chaperonin 10 - genetics; Chaperonin 60 - biosynthesis; Chaperonin 60 - genetics; Cloning, Molecular; Culture Media, Conditioned; Deoxyadenine Nucleotides - analysis; Deoxyadenine Nucleotides - chemistry; Deoxycytosine Nucleotides - analysis; Deoxycytosine Nucleotides - chemistry; Dimerization; Enzyme Inhibitors - chemistry; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - growth & development; Ethanol - chemistry; Humans; Isoenzymes - antagonists & inhibitors; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - isolation & purification; Kinetics; Models, Molecular; Recombinant Fusion Proteins - antagonists & inhibitors; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Solubility; Spectrometry, Fluorescence; Substrate Specificity; Thymidine Kinase - antagonists & inhibitors; Thymidine Kinase - chemistry; Thymidine Kinase - genetics; Thymidine Kinase - isolation & purification; Thymine Nucleotides - analysis; Thymine Nucleotides - chemistry; Tryptophan - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi035230f

Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 °C minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least ∼5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.