Reaction Mechanism of 21S Dynein ATPase from Sea Urchin Sperm. II. Formation of Reaction Intermediates1

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Abstract
The amounts of ATP and ADP bound to 21S dynein during the ATPase reaction were measured in the presence of 2.83 mg/ml 21S dynein, 2 mM PEP, 4 mg/ml PK, 0.1 M KCl, 5 mM MgCl 2 1 mM DTT, 0.1 mM PMSF, 50% [2- 3 H]glycerol and 20 mM imidazole at pH 7.0 and 0°C. The maximum amounts of ATP and ADP bound to 21S dynein were 0.29 and 0.55 mol/(10 6 g protein), respectively. The dissociation constants of ATP for the ATP and ADP binding (4 μM) were almost equal to the Km value (3.7 μM) of dynein-ATPase in the steady state. The amount of bound ADP during the initial phase showed an overshoot, which reached 0.6–0.8 mol/10 6 g protein at 5 s, then decreased to the steady state level within 20 s. Furthermore, the rate of TCA-P 1 liberation during the initial 5 s was 6 times the steady-state rate. The apparent P 1 -burst size, estimated by extrapolating the steady-state P 1 liberation to zero time, was 1.33 mol/(10 6 g protein). The true P 1 size was calculated to be 1.56 mol/(10 6 g protein) by correcting for the effect of P 1 liberation at steady state. All these findings could be explained quantitatively by the following reaction scheme for 21 S dynein ATPase in the presence of glycerol: where K 1 =25.5 μM and k2 k 3 and k 4 were 0.39, 0.21, and 0.11 s −1 , respectively.