FIG. 5.

Phosphoryl transfer from HPr∼P of S. thermophilus, B. subtilis, and E. coli to IIA^LacS-6H and IIA^LacS-6H(I548E/G556D). (A) Time course of the phosphoryl transfer reaction between 25 μM HPr∼P from S. thermophilus and IIA^LacS-6H. (B) Phosphorylation rate of IIA^LacS-6H by HPr∼P from S. thermophilus (•), B. subtilis, (■), and E. coli (▴). (C) Phosphorylation rate of IIA^LacS-6H(I548E/G556D) by HPr∼P from S. thermophilus (•), B. subtilis (■), and E. coli (▴). The phosphorylation reaction was started by adding 5.8 μM purified IIA^LacS-6H or 4.1 μM IIA^LacS-6H(I548E/G556D) to 50 mM Tris acetate (pH 7.5) containing 1 mM DTT, 2 mM MgCl₂, 0.8 μM purified enzyme I, 10 mM PEP, and HPr at concentrations ranging from 0 to 90 μM, in a total volume of 10 μl at 10°C. The reactions were stopped by addition of 10 μl of 2× SDS sample buffer.