SEPARATION OF L-PRO-DL-BOROPRO INTO ITS COMPONENT DIASTEREOMERS AND KINETIC-ANALYSIS OF THEIR INHIBITION OF DIPEPTIDYL PEPTIDASE .4. A NEW METHOD FOR THE ANALYSIS OF SLOW, TIGHT-BINDING INHIBITION

The potent dipeptidyl peptidase IV (DP IV) inhibitor [1-(2-pyrrolidinylcarbonyl)-2-pyrrolidinyl]boronic acid (L-Pro-DL-boroPro) [Flentke, G. R., Munoz, E., Huber, B. T., Plaut, A. G., Kettner, C. A., & Bachovchin, W. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,1556-1559] was fractionated into its component L-L and L-D diastereomers by C18 HPLC, and the binding of the purified diastereomers to DP IV was analyzed. Inhibition kinetics confirms that the L-L diastereomer is a potent inhibitor of DP IV, having a K(i) of 16 pM. The L-D isomer binds at least 1000-fold more weakly than the L-L, if it binds at all, as the approximately 200-fold weaker inhibition observed for the purified L-D isomer is shown here to be due entirely to the presence of a small amount (0.59%) of the L-L diastereomer contaminating the L-D preparation. The instability of Pro-boroPro, together with its very high affinity for DP IV and the time dependence of the inhibition, makes a rigorous kinetic analysis of its binding to DP IV difficult. Here we have developed a method which takes advantage of the slow rate at which the inhibitor dissociates from the enzyme. The method involves preincubating the enzyme and the inhibitor without substrate and then assaying the free enzyme by the addition of substrate and following its hydrolysis for a period of time which is short relative to the dissociation rate of the inhibitor. Data from experiments in which the preincubation time was sufficient for enzyme and inhibitor to reach equilibrium were analyzed by fitting to an appropriate form of the quadratic equation and yielded a K(i) value of 16 pM. Data from experiments in which the incubation time was insufficient to establish equilibrium, i.e., within the slow-binding regime, were analyzed by fitting to an integrated rate equation. The appropriate integrated rate equation for an A + B reversible C system going to equilibrium does not appear to have been previously derived. The analysis of the slow-binding curves yielded a K(i) value of 16 pM, in agreement with that of 16 pM determined in the equilibrium titrations, and a bimolecular rate constant of association, k(on), of 5.0 x 10(6) M-1 s-1. The experimentally determined k(on) and K(i) indicate that the dissociation rate constant, k(off), is 78 x 10(-6) s-1 (t1/2 = 150 min). The slow-binding curves are shown here to fit a simple E + I reversible EI model, indicating that it is not necessary to invoke a two-step mechanism to explain the inhibition kinetics.