Kinetic Analysis and Ligand-Induced Conformational Changes in Dimeric and Tetrameric Forms of Human Thymidine Kinase 2

Abstract

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.

Structure-guided Engineering of Human Thymidine Kinase 2 as a Positron Emission Tomography Reporter Gene for Enhanced Phosphorylation of Non-natural Thymidine Analog Reporter Probe

“…Data were fit to the Michaelis-Menten equation using SigmaPlot. Of note, in some previous reports, negative cooperativity was observed with thymidine but not with deoxycytidine (32,33). When we fit our data for WT TK2 using the Hill equation, we also see the same magnitude of negative cooperativity as reported by others (n ϭ ϳ0.7) with thymidine and the analogs tested.…”

Thermostability and molecular encapsulation within an engineered caged protein scaffold

“…The solubility of the protein was used to generate the initial thermostability profile. Multicomponent protein assemblies often exhibit irreversible unfolding upon heating (Backmann et al, 1998;Barroso et al, 2005;Blaauwen and Driessen, 1998), and we observe this as well when our system is heated. As presented in Figure 5A, heating the protein for 20 min at elevated temperatures does not affect protein solubility until the regime between 75 and 958C.…”

Mechanisms of Hepatocellular Dysfunction and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration

“…Thymidine kinase is a TTP insensitive dimeric form (one catalytic site and other allosteric site) means K m for enzyme decreases with elevated ATP concentration (ATP positive effector and TTP negative effector). dimeric form and shows sigmoid behavior of thymidine kinase [Barroso et al 2005] cultures added with trophozoits, nitroimidazole, exhibited increased protein degradation. Lysosome as proteolytic component was shown to contribute for protein degradation as earlier various reports indicated protein size dependence [Neff et al1990].…”