SLOW TIGHT-BINDING INHIBITION OF PROLYL ENDOPEPTIDASE BY BENZYLOXYCARBONYL-PROLYL-PROLINAL

Prolyl endopeptidase is a serine proteinase that specifically cleaves peptides on the carboxy side of proline residues. Wilk and Orlowski [(1983) J. Neurochem. 41, 69-75] have shown that benzyloxycarbonyl-prolyl-prolinal (Z-prolyl-prolinal) is a potent inhibitor of prolyl endopeptidase. We show that Z-prolyl-prolinal is a slow-binding inhibitor of mouse brain prolyl endopeptidase with K(i) 0.35 ± 0.05 nM. Kinetic analysis indicates that the mechanism is a simple, but slow, reversible equilibrium between free and bound enzyme (E + I ⇆ EI) with rate constants for association (k(on)) and dissociation (k(off)) of 1.6 x 105 M-1 · s-1 and approx. 4 x 10-5 s-1 respectively. Slow-binding inhibition is dependent on the presence of the aldehyde group since the alcohol (Z-prolyl-prolinol) is a rapid and 50,000-fold poorer inhibitor (K(i) 19 μM). Prolyl endopeptidase from human brain is also inhibited by Z-prolyl-prolinal with kinetics similar to those of the mouse brain enzyme.