PYRUVATE-EXTENDED AMINO-ACID DERIVATIVES AS HIGHLY POTENT INHIBITORS OF CARBOXYL-TERMINAL PEPTIDE AMIDATION

Carboxyl-terminal amidation, a required post-translational modificatio n for the bioactivation of many neuropeptides, entails sequential enzy matic action by pep tidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The mo nooxygenase, P AM, first catalyzes conversion of a glycine-extended pro-peptide to th e corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, t hen catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We now introduce the first potent i nhibitors for peptidylamidoglycolate lyase. These inhibitors, which ca n be viewed as pyruvate extended N-acetyl amino acids, constitute a no vel class of compounds. They were designed to resemble likely transien t species along the reaction pathway of PGL catalysis. A general synth etic procedure for preparation of pyruvate-extended N-acetyl amino aci ds or peptides is described. Since these compounds possess the 2,4-dio xo-carboxylate moiety, their solution tautomerization was investigated using both NMR and high performance liquid chromatography analyses. T he results establish that freshly prepared solutions of N-Ac-Phe-pyruv ate consist predominantly of the enol tautomer, which then slowly taut omerizes to the diketo form when left standing for several days in an aqueous medium; upon acidification, formation of the hydrate tautomer occurs. Kinetic experiments established that these novel compounds are highly potent, pure competitive inhibitors of PGL. Kinetic experiment s with the ascorbate dependent copper monooxygenases, PAM and dopamine -beta-monooxygenase, established that these compounds also bind compet itively with respect to ascorbate; however, pyruvate-extended N-acyl-a mino acid derivatives possessing hydrophobic side chains are much more potent inhibitors of PGL than of PAM. Selective targeting of N-Ac-Phe -pyruvate so as to inhibit the lyase, but not the monooxygenase, domai n was demonstrated with the bifunctional amidating enzyme of Xenopus l aevis. The availability of potent inhibitors of PGL should facilitate studies regarding the possible biological role of alpha-hydroxyglycine -extended peptides.