Figure 3. Catalytic effect of the Mg2+ cofactor.

Pre-steady-state kinetics of EAdk at 25 °C measured by quench-flow (a) with and (b,c) without Mg2+ (n = 3 experiments; mean ± s.e.m.). (a) 113 μM EAdk, 4 mM ATP, 4 mM AMP and 8 mM MgCl2 in the forward direction (green) and 4 mM ADP and MgCl2 in the reverse direction (blue) were used. (b) A resolved burst phase is seen in the forward direction with 20 μM EAdk and 4 mM ATP and AMP, providing the rates of both the P-transfer and lid-opening, while in the reverse reaction (c) No burst phase is seen with 20 μM or 500 μM EAdk (inset) and 4 mM ADP.