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Forskningsartikel2018Vetenskapligt granskadÖppen tillgång

Negative Cooperative Binding of Thymidine, Ordered Substrate Binding, and Product Release of Human Mitochondrial Thymidine Kinase 2 Explain Its Complex Kinetic Properties and Physiological Functions

Wang, Liya; Zhang, Li; Sun, Ren; Eriksson, Staffan


Mitochondrial thymidine kinase 2 (TK2) catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC) and is essential for mitochondrial function in post-mitotic tissues. The phosphorylation of dT shows negative cooperativity, but the phosphorylation of dC follows classical Michaelis-Menten kinetics. The enzyme is feedback-inhibited by its end products deoxythymidine triphosphate (dTTP.)and deoxycytidine triphosphate (dCTP). In order to better understand the reaction mechanism, and the negative cooperative behavior, we conducted isothermal titration calorimetry (ITC) and intrinsic tryptophan fluorescence (ITF) quenching studies with purified recombinant human TK2. Cooperative binding was observed with dT but not dC by the ITC analysis in accordance with earlier enzyme kinetic studies. The phosphate donor adenosine triphosphate (ATP) did not bind to either dTTP-bound or dTTP-free enzymes but bound tightly to the dT- or dC-TK2 complexes with large differences in enthalpy and entropy changes, strongly suggesting an ordered binding of the substrates and different conformational states of the ATP and dT- and dC-TK2 ternary complexes. dTTP binding was endothermic; however, dCTP could not be shown to interact with the enzyme. ITF quenching studies also revealed tight binding of dT, dC, deoxythymidine monophosphate, deoxycytidine monophosphate, and dTTP but not adenosine 5'-diphosphate or ATP. These results strongly indicate an ordered sequential binding of the substrates and ordered release of the products as well as different conformational states of the active site of TK2. These results help to explain the different kinetics observed with dT and dC as substrates which have important implications for TK2 regulation in vivo.

Publicerad i

ACS Omega
2018, Volym: 3, nummer: 8, sidor: 8971-8979