Inhibition of Rabbit Brain Prolyl Endopeptidase by N‐Benzyloxycarbonyl‐Prolyl‐Prolinal, a Transition State Aldehyde Inhibitor

Abstract: Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin‐releasing hormone, and luteinizing hormone‐releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl‐prolyl‐proline, with a K_i of 50 μM, prompted the synthesis of benzyloxycar‐bonyl‐prolyl‐prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a K_i of 14 nM. This K_i is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S₁ subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the K_i for prolyl endopeptidase. The data indicate that benzyloxycarbonyl‐prolyl‐prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.