Aminoketone 1, a ~F[CO-CH] dipeptide mimic of glycylproline, is a potent, competitive inhibitor (Ki = 270 + 24 nM) of porcine kidney prolidase, a Mn(II)-dependent dipeptidase, whereas the homologous aminoketone 2 and its non-cyclic analogue 8-amino-levulinic acid, 3, are significantly less inhibitory (Ki = 1 mM).Prolidase (EC 3.4.13.9), discovered in 1937,1 is a manganese-dependent dipeptidase which hydrolyzes glycylproline to glycine and proline (see Scheme I). 2 The primary physiological role of prolidase is thought to be the recycling of proline in the metabolism of collagen. 3 Prolidase activity has been detected in the brain, 4 implicating this activity in the regulation of the concentration of proline, which is demonstrably neuroactive, in the brain tissue. 4d In humans, a genetic deficiency of prolidase results in a complex clinical syndrome which includes chronic ulcerative dermatitis and mental retardation. 5Though the precise mechanism by which prolidase effects peptide bond hydrolysis is not clear, a proposed mechanism has been outlined by Mock and Liu. 6 Prolidase h~Cdrolyzes the transoid peptide bond specifically 7 and several inhibitors have been reported which are competitive with respect to the Gly-Pro substrate. 8 Perhaps most intriguing is the observation of Radzicka and Wolfenden 9 that phosphoenol pyruvate (PEP) strongly inhibits this enzyme at concentrations which approximate the intracellular concentration of PEP. Mock and Liu t0 have recently shown that inhibition of prolidase by PEP is biphasic and they postulated a negative cooperativity between the enzyme monomers, so as to avoid inactivation of prolidase by extant PEP.We have prepared a bisubstrate analogue of Gly-Pro in which the pyrrolidine nitrogen atom has been replaced by a methine carbon to give cyclopentane 1, as well as its homologue 2. Similar ketomethylene isosteres have been used as hydrolysis-resistant inhibitors of peptidases and proteases. 11 For example, tripeptide Bz-Phe-Gly-Pro is an inhibitor (IC50 = 9.4 ~M) of angiotensin converting enzyme, but its ketomethylene analogue (Bz-Phe-Ud[CO-CH2]-Gly-Pro) was significantly more potent (IC50 = 0.07 ~flVI). 12 Below we describe the syntheses and inhibition constants of aminoketones 1, 2, and 3 (iS-aminolevulinic acid) against porcine kidney prolidase.