Separation of L-Pro-DL-boroPro into its component diastereomers and kinetic analysis of their inhibition of dipeptidyl peptidase IV. 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 Ki 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.(ABSTRACT TRUNCATED AT 250 WORDS)