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. 2012 May 27;287(30):24894–24904. doi: 10.1074/jbc.M112.376830

FIGURE 3.

FIGURE 3.

Kinetics of ATP hydrolysis by acid quench. The MT·CENP-E complex was rapidly mixed with increasing concentrations of [α-32P]ATP in a chemical quench-flow instrument for 5–300 ms followed by a formic acid quench to terminate the reaction. Final concentrations: 5 μm CENP-E, 15 μm MTs, 5–200 μm [α-32P]ATP, 100 mm KCl. A, representative transients show an exponential increase of [α-32P]ADP·Pi product formation during the first ATP turnover followed by a linear phase of product formation. B, the observed exponential burst rates were plotted as a function of ATP concentration, and the hyperbolic fit of the data provided k+2 = 24.7 ± 0.5 s−1 with Kd,ATP = 12.3 ± 1.1 μm. C, the amplitudes of the pre-steady-state exponential burst phase were plotted as a function of ATP concentration, and the fit of the data yields Amax = 1.93 ± 0.1 ADP·Pi/site with apparent Kd,ATP = 44.8 ± 4.4 μm. Data shown in panels B and C are from multiple experiments.