Table 2.
Experimental Conditions for Hypoxanthine and Xanthine Kinetics Determinations.
enzyme | [Hypoxanthine] μM | [Xanthine] μM | Time Courses (both substrates) |
---|---|---|---|
htTGT(wt) | 25–3000 | 10–1500 | 120 min |
htTGT(D143A) | 50–2000 | 1–750 | 120 min |
htTGT(D143N) | 50–2000 | 0.5–750 | 120 min |
htTGT(D143S) | 1–3000 | 1–3000 | 4 hr |
htTGT(D143T) | 1–3000 | 1–3000 | 4 hr |
[tRNA] = 20 μM for all enzymes and [TGT] = 100 nM for all except htTGT(D143S) & htTGT(D143T) = 250 nM. Assays were conducted in 100 mM HEPES (pH 7.3), 20 mM MgCl2 and 5 mM DTT at 37 °C as described in Methods.