Avidin is a slow-binding inhibitor of pyruvate carboxylase

In the presence of avidin, progress curves for the pyruvate carboxylase (EC 6.4.1.1) reaction show a marked decrease in the rate of reaction over a period of several minutes. These curves are quantitatively described by an equation in which the rate of catalysis by the enzyme undergoes a firstorder decay from a high initial velocity to a final velocity which is close to zero. The initial velocity of the reaction is independent of avidin concentration while the decay constant increases linearly with increasing concentrations of avidin. These findings are in agreement with a model in which there is slow equilibration between free enzyme and the enzyme-avidin complex. Alternative models involving slow isomerization of the free enzyme or of an enzyme-avidin complex are not consistent with the data. From the slope of the decay constant vs. avidin concentration plot, the rate constant for formation of the enzyme-avidin complex was calculated to be (1.42 ± 0.09) X 105 M-1 s-1 for the enzyme isolated from chicken liver. The intercept of this plot, which is equal to the rate constant for dissociation of the enzyme-avidin complex, was not well-defined by the data but was estimated to be less than 2 X 10-3 s-1. In view of this uncertainty, an alternative procedure was used to obtain a more reliable value. This procedure involved following the regeneration of enzymic activity upon incubation of the enzyme-avidin complex with biotin and yielded a value of (1.45 ± 0.13) X 10-5 s-1 for the dissociation rate constant. The enzyme-avidin complex undergoes a reaction which renders the inhibition of the enzyme irreversible and which occurs at a rate that is 5 times greater than the rate of dissociation of the avidin from the enzyme. This observation accounts for the widely held view that avidin inhibition is irreversible.