Figure 7.

Direct measurement of Pi release from Gle1–Dbp5 via P_iBP. (A) Time courses of phosphate release in a pre-equilibrated mixture of 1 μM Dbp5 (0.5 μM after mixing) and 20 μM Gle1 (10 μM after mixing) upon rapid mixing with various [ATP] (0, 5, 10, 15, 20, 50, 100 μM after mixing) containing 6 μM P_iBP (3 μM after mixing). Continuous lines through the data are the best fits to a linear equation. (B) [ATP]-dependance of the observed steady-state P_i release in A (solid squares) or steady-state ATP hydrolysis in the presence of the NADH regenerating system (26,31,49). Continuous lines through the data are the best fits to a rectangular hyperbola yielding the maximum velocity per enzyme (k_cat= 0.15 ± 0.02 s^–1 Dbp5^–1_, circles; k_cat= 0.12 ± 0.01 s^–1 Dbp5^–1_, squares) from the amplitude and K_M (20 ± 3 μM, circles; 26 ± 6 μM, squares) from the [ATP] at half-maximum velocity (Table 1). Uncertainty bars represent standard errors in the fits and are contained within the data points. InsP₆ is included in all experiments at an equimolar concentration with Gle1.