Pyruvate-extended amino acid derivatives as highly potent inhibitors of carboxyl-terminal peptide amidation.
Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We now introduce the first potent inhibitors for peptidylamidoglycolate lyase. These inhibitors, which can be viewed as pyruvate-extended N-acetyl amino acids, constitute a novel class of compounds. They were designed to resemble likely transient species along the reaction pathway of PGL catalysis. A general synthetic procedure for preparation of pyruvate-extended N-acetyl amino acids or peptides is described. Since these compounds possess the 2,4-dioxo-carboxylate moiety, their solution tautomerization was investigated using both NMR and high performance liquid chromatography analyses. The results establish that freshly prepared solutions of N-Ac-Phe-pyruvate consist predominantly of the enol tautomer, which then slowly tautomerizes 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 experiments with the ascorbate-dependent copper monooxygenases, PAM and dopamine-beta-monooxygenase, established that these compounds also bind competitively with respect to ascorbate; however, pyruvate-extended N-acyl-amino 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, domain was demonstrated with the bifunctional amidating enzyme of Xenopus laevis. The availability of potent inhibitors of PGL should facilitate studies regarding the possible biological role of alpha-hydroxyglycine-extended peptides.
Mounier
C E
CE
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Shi
J
J
Sirimanne
S R
SR
Chen
B H
BH
Moore
A B
AB
Gill-Woznichak
M M
MM
Ping
D
D
May
S W
SW
D000577:Amides / Q000378:metabolism*; D019760:Amidine-Lyases; D000818:Animals; D002417:Cattle; D008190:Lyases / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009097:Multienzyme Complexes; D010455:Peptides / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism*; D014982:Xenopus laevis
D000577:Amides; D009097:Multienzyme Complexes; D010455:Peptides; D019289:Pyruvic Acid; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D008190:Lyases; D019760:Amidine-Lyases; C068521:peptidylamidoglycolate lyase