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

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 K_m 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.