ADP release is rate limiting in steady-state turnover by the dynein adenosinetriphosphatase.
The kinetics of the product release steps in the pathway of ATP hydrolysis by dynein were investigated by examining the rate and partition coefficient of phosphate-water 18O exchange under equilibrium and steady-state conditions. Dynein catalyzed both medium and intermediate phosphate-water oxygen exchange with a partition coefficient of 0.30. The dependence of the rate of loss of the fully labeled phosphate species on the concentration of ADP was hyperbolic, with an apparent Kd for the binding of ADP to dynein of 0.085 mM. The apparent second-order rate constant for phosphate binding to the dynein-ADP complex was 8000 M-1 s-1. The time course of medium phosphate-water oxygen exchange during net ATP hydrolysis was examined in the presence of an ATP regeneration system. The observed rate of loss of P18O4 was comparable to the rate observed at saturating ADP which implies that ADP release is rate limiting for dynein in the steady state. Product inhibition of the dynein ATPase was also examined. ADP inhibited the enzyme competitively with a Ki of 0.4 mM. Phosphate was a linear noncompetitive mixed-type inhibitor with a Ki of 11 mM. These data were fit to a model in which phosphate release is fast and is followed by rate-limiting release of ADP, allowing us to define each rate constant in the pathway. A discrepancy between the total free energy calculated compared to the known free energy of ATP hydrolysis suggests that there is an additional step in the pathway, perhaps involving a change in conformation of the enzyme-ADP state preceding ADP release.
Holzbaur
E L
EL
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Johnson
K A
KA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004398:Dyneins; D005033:Ethylmaleimide / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism; D010103:Oxygen Isotopes; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D013768:Tetrahymena / Q000201:enzymology
D015879:Myosin Subfragments; D010103:Oxygen Isotopes; D010446:Peptide Fragments; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D004398:Dyneins; D005033:Ethylmaleimide