The kinetic effects on thymidine kinase 2 by enzyme-bound dTTP may explain the mitochondrial side effects of antiviral thymidine analogs

Abstract

Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in the salvage of pyrimidine deoxynucleosides needed for mitochondrial DNA synthesis. TK2 phosphorylates thymidine (dThd), deoxycytidine (dCyd), and many other antiviral pyrimidine nucleoside analogs. Zidovudine (AZT) is the first nucleoside analog approved for anti-HIV therapy, and it is still used in combination with other drugs. One of the side effects of long-term treatment with nucleoside analogs is mitochondrial DNA depletion, which has been ascribed to competition by AZT for the endogenous dThd phosphorylation carried out by TK2. Here we studied the kinetics of AZT and 3'-fluorothymidine phosphorylation by recombinant human TK2 and the effects of these and other pyrimidine nucleoside analogs on the phosphorylation of dThd and dCyd. Thymidine analogs strongly inhibited dThd phosphorylation but not dCyd phosphorylation, which instead was stimulated ∼30%. We found that recombinant human TK2 contained the feedback inhibitor dTTP in a 1:1 molar ratio and that incubation with dThd and AZT could completely remove the enzyme-bound dTTP, but dCyd was less efficient in this regard. The release of feedback inhibitor by dThd and dThd analogs most likely accounts for the observed kinetics. Similar effects were also observed with native rat liver mitochondrial TK2, strongly indicating a physiologic role for this process, which most likely is an important factor in the mitochondrial toxicity observed with antiviral nucleoside analogs.

Figures

Fig. 1.

Characterization of recombinant human TK2 with AZT and FLT. (A) Substrate saturation curve of AZT (■) and FLT (●). (B) Dixon plots of 1/v versus AZT concentration. The concentrations of dThd were 0.2 μM (●), 0.5 μM (▴), and 1.0 μM (■). (C) Dixon plots of 1/v versus FLT concentration. The concentrations of dThd were 0.2 μM (●) and 0.6 μM (■). (D) Stimulation of dCyd phosphorylation by AZT. dCyd concentrations were 0.2, 0.5, and 1.0 μM. (E) Stimulation of dCyd phosphorylation by FLT. dCyd concentrations were 0.2, 0.5, and 1.0 μM. (F) Plots of 1/v versus AZT concentration (at fixed concentrations of FLT of 0, 1.2, 2.4, 4.8, and 6.0 μM). The concentrations of dThd and ATP were 0.6 μM and 2 mM, respectively. All measurements were repeated at least 3 times, and data are plotted as means ± standard errors of the means (error bars).

Fig. 2.

Characterization of rat liver mitochondrial TK2. (A) SDS-PAGE analysis of purified native rat liver mitochondrial TK2 (lane 1, the arrow indicates the TK2 band) and recombinant human TK2 (lane 2); (B) substrate saturation curves (■, dThd; ●, dCyd); (C) inhibition of dThd phosphorylation by AZT (■) and FLT (●); (D) stimulation of dCyd phosphorylation by AZT and FLT. All measurements were repeated at least 3 times, and data are plotted as mean ± standard errors of the means (error bars).

Fig. 3.

HPLC analysis of enzyme-bound feedback inhibitor. (A) HPLC standards; (B) recombinant human TK2; (C) recombinant human TK2 (20 μM) incubated with 200 μM dThd or AZT; (D) recombinant human TK2 incubated with dCyd. After dialysis, the protein was precipitated with 10% PCA and removed by centrifugation, and the supernatant was analyzed by HPLC after neutralization. Abs, absorbance.

Fig. 4.

Effects of AZT and FLT on phosphorylation of dThd and dCyd by dTTP-free recombinant human TK2. Data were from 3 independent measurements and are plotted as means ± standard errors of the means (error bars).