Kinetics of vanadate dissociation: estimation of the rate by inhibitor inactivation

Vanadate (+5) is a potent inhibitor of a variety of ATPases including dynein ATPase. We describe a method useful for estimating the functional dissociation rate of vanadate from the active site which does not rely on classical physical separation techniques. The method involves spectrophotometrically monitoring the enzymatic activity as the inhibitor dissociates from the enzyme and is inactivated by norepinephrine. Norepinephrine effectively reverses vanadate inhibition by reducing vanadate (+5) to oxovanadium (+4). This reduction by norepinephrine is sufficiently fast for these purposes--addition of vanadate after norepinephrine shows no inhibition of ATPase activity. The mathematical estimation procedure is generally useful for estimation of dissociation rates of other reversible inhibitors which can be quickly inactivated after dissociation from the enzyme. The rate of dissociation of vanadate from dynein with ATP and 2-N3ATP as substrates using this method was estimated to be in the ranges 0.0023-0.0042 and 0.0057-0.0075 s-1, respectively. These rates permit estimation of the rates of vanadate association with dynein by using the reported dissociation constant for vanadate. The results are consistent with the very fast and potent inhibition of dynein ATPase activity observed.