FIGURE 4.

Phosphoenzyme formation by utilization of ATP. A, the reaction was started by addition of 25 μm [γ-³²P]ATP to WT CopA in the presence of 1 mm BCS (absence of Cu⁺) (•), with 40 μm CuCl₂ added in conjunction with [γ-³²P]ATP (○), or by addition of [γ-³²P]ATP following a 2-min preincubation with 40 μm Cu²⁺ (▾). Temperature was 60 °C. B, initial velocity of phosphoenzyme formation as a function of ATP concentration. CopA was preincubated for 2 min with 1 mm BCS before addition of [γ-³²P]ATP. Temperature was 40 °C. See “Materials and Methods” for reaction mixtures.