Presteady state kinetic analysis of vanadate-induced inhibition of the dynein ATPase.
The effects of vanadate on the kinetics of ATP binding and hydrolysis by Tetrahymena 30 S dynein were examined by presteady state kinetic analysis. Up to a concentration of 400 microM, vanadate did not inhibit the rate or amplitude of the ATP binding-induced dissociation of the microtubule-dynein complex measured by stopped flow light-scattering methods. Chemical quench flow experiments showed that vanadate (80 microM) did not alter the rate or amplitude of the presteady state ATP binding or ATP hydrolysis transients, but the steady state hydrolysis of ATP was blocked immediately after a single turnover of ATP. Preincubation of the enzyme with ADP and vanadate inhibited both presteady state and steady state hydrolysis. These data suggest that vanadate acts as a phosphate analog to form an enzyme-ADP-vanadate complex, analogous to the transition state during catalysis, by the following pathway: (formula; see text) where V represents vanadate and D represents a dynein active site. ADP and vanadate, added together, induced dissociation of the microtubule-dynein complex at a maximum rate of 0.6 S-1. These observations imply that a microtubule-dynein-ADP-vanadate complex was formed which subsequently dissociated as shown below: (formula; see text) where M denotes a microtubule. The ADP plus vanadate-induced dissociation may represent the reverse of the normal forward pathway involving the binding of a dynein-ADP-phosphate complex to a microtubule.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000818:Animals; D004398:Dyneins / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008433:Mathematics; D008870:Microtubules / Q000201:enzymology; D008954:Models, Biological; D010761:Phosphorus Radioisotopes; D013768:Tetrahymena / Q000201:enzymology*; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D010761:Phosphorus Radioisotopes; D014639:Vanadium; D014638:Vanadates; D000251:Adenosine Triphosphatases; D004398:Dyneins