Kinetic analysis and ligand-induced conformational changes in dimeric and tetrameric forms of human thymidine kinase 2.
Recombinant human thymidine kinase 2 (hTK2) expressed in Escherichia coli has been found to bind tightly a substoichiometric amount of deoxyribonucleoside triphosphates (dTTP > dCTP >> dATP), known to be strong feedback inhibitors of the enzyme. Incubation of hTK2 with the substrate dThd was able to release the dNTPs from the active site during purification from E. coli and thus allowed the kinetic characterization of the noninhibited enzyme, with the tetrameric hTK2 showing slightly higher activity than the most abundant dimeric form. The unliganded hTK2 revealed a lower structural stability than the inhibitor-bound enzyme forms, being more prone to aggregation, thermal denaturation, and limited proteolysis. Moreover, intrinsic tryptophan fluorescence (ITF), far-UV circular dichroism (CD), and limited proteolysis have revealed that hTK2 undergoes distinct conformational changes upon binding different substrates and inhibitors, which are known to occur in the nucleoside monophosphate kinase family. The CD-monitored thermal denaturation of hTK2 dimer/tetramer revealed an irreversible process that can be satisfactorily described by the two-state irreversible denaturation model. On the basis of this model, the parameters of the Arrhenius equation were calculated, providing evidence for a significant structural stabilization of the enzyme upon ligand binding (dCyd < MgdCTP < dThd < dCTP < dTTP < MgdTTP), whereas MgATP further destabilizes the enzyme. Finally, surface plasmon resonance (SPR) was used to study in real time the reversible binding of substrates and inhibitors to the immobilized enzyme. The binding affinities for the inhibitors were found to be 1-2 orders of magnitude higher than for the corresponding substrates, both by SPR and ITF analysis.
Barroso
João Filipe
JF
Department of Biomedicine, Section of Biochemistry and Molecular Biology, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway.
Carvalho
Raquel Negrão
RN
Flatmark
Torgeir
T
D002942:Circular Dichroism / Q000379:methods; D003854:Deoxyribonucleotides / Q000378:metabolism; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance / Q000379:methods; D013937:Thymidine Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D003854:Deoxyribonucleotides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008024:Ligands; D011994:Recombinant Proteins; D014364:Tryptophan; C105339:thymidine kinase 2; D013937:Thymidine Kinase; D014357:Trypsin