Inhibition of Peptidylglycine .alpha.-Amidating Monooxygenase by N-Substituted Homocysteine Analogs

Erion, Mark D.; Tan, Jenny; Wong, M.; Jeng, Arco Y.

doi:10.1021/jm00052a002

Cited by 24 publications

(32 citation statements)

References 18 publications

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“…These include substrate-analog competitive inhibitors, i.e. O-glycolate esters (11,13) and homocysteine-terminating peptides (14), as well as olefinic mechanism-based inactivators (13,15,16). All of these compounds are inhibitors of the PAM-catalyzed monooxygenation step in the amidation process.…”

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confidence: 99%

Pyruvate-extended Amino Acid Derivatives as Highly Potent Inhibitors of Carboxyl-terminal Peptide Amidation

Mounier

Shi

Sirimanne

et al. 1997

Journal of Biological Chemistry

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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 ␣-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this ␣-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-AcPhe-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-␤-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 ␣-hydroxyglycine-extended peptides.Neuropeptides, which are critical mediators of intercellular communication, are generated biosynthetically from larger precursors via a variety of post-translational modifications. One such processing event is carboxyl-terminal amidation, a very prevalent post-translational modification essential to the bioactivity of many neuropeptides (1, 2). We and others (3-8) have demonstrated that formation of peptide amides from their glycine-extended precursors is a two-step process, entailing sequential enzymatic action by peptidylglycine monooxygenase (PAM, 1 EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes formation of the ␣-hydroxyglycine derivative of the glycine-extended precursor, in a process dependent upon ascorbate, copper, and molecular oxygen (3, 4, 9). The lyase, PGL, then catalyzes the breakdown of this ␣-hydroxyglycine de...

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confidence: 99%

Pyruvate-extended Amino Acid Derivatives as Highly Potent Inhibitors of Carboxyl-terminal Peptide Amidation

Mounier

Shi

Sirimanne

et al. 1997

Journal of Biological Chemistry

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“…Chemical Syntheses-Hydrocinnamoyl-L-phenylalanyl-L-homocysteine (Compound 1) was synthesized as described previously (30). A general procedure for the synthesis of prodrug esters of Compound 1 is shown in Scheme 1, using hydrocinnamoyl-L-phenylalanyl-D,L-homocysteine thiolactone (Compound 2) as a common intermediate.…”

Section: Materials-n-mentioning

confidence: 99%

“…PAM Enzyme Assay-PAM was partially purified from conditioned medium of cultured rat medullary thyroid carcinoma CA-77 cells by DEAE and Sephacryl 300 SF column chromatography (12). The enzyme assay was performed according to the method published previously (30). Briefly, PAM (13.5 milliunits) was pre-incubated with various concentrations of inhibitors in 150 mM Tes, pH 7.0, and 0.001% Triton X-100 for 20 min at room temperature in a total volume of 50 l. An equal volume of a solution containing 4 M substrate, N-dansyl-D-Tyr-PheGly, and 6 mM ascorbate was added and incubated further for 20 min.…”

Section: Inhibition Of Sp Biosynthesis By Esters Of Pam Inhibitorsmentioning

confidence: 99%

“…Studies in cultured dorsal root ganglion (DRG) cells, however, showed only modest inhibition of substance P biosynthesis with these compounds (30). Since PAM is localized in secretory granules, these results suggest that the inhibitors were not accessible to the intracellular compartment of the cells.…”

mentioning

confidence: 93%

“…In a previous study, N-substituted homocysteine analogs were found to be potent inhibitors of PAM partially purified from conditioned medium of cultured rat medullary thyroid carcinoma CA-77 cells (30). Studies in cultured dorsal root ganglion (DRG) cells, however, showed only modest inhibition of substance P biosynthesis with these compounds (30).…”

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confidence: 97%

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Suppression of Substance P Biosynthesis in Sensory Neurons of Dorsal Root Ganglion by Prodrug Esters of Potent Peptidylglycine α-Amidating Monooxygenase Inhibitors

Jeng

Fujimoto

Chou

et al. 1997

Journal of Biological Chemistry

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Substance P as well as many other neuropeptides are synthesized as glycine-extended precursors and converted to the biologically active C-terminal amides by posttranslational modification. The final step of posttranslational processing is catalyzed by peptidylglycine ␣-amidating monooxygenase (PAM). In a previous study, N-substituted homocysteine analogs were found to be potent inhibitors of PAM partially purified from conditioned medium of cultured rat medullary thyroid carcinoma CA-77 cells. These compounds, however, were only modest inhibitors of substance P production in cultured dorsal root ganglion cells, possibly because of poor cell penetration. Several ester derivatives of hydrocinnamoyl-phenylalanyl-homocysteine, one of the most potent PAM inhibitors, were prepared to increase the intracellular accessibility of these compounds. Hydrocinnamoyl-phenylalanyl-(S-benzoyl-homocysteine) benzyl ester was identified as the most potent compound, inhibiting substance P biosynthesis in dorsal root ganglion cells with an IC 50 of 2 M. Inhibition of PAM resulted in a concomitant increase in the glycine-extended substance p (substance P-Gly) precursor peptide. In the presence of 3 M benzyl ester derivative, the intracellular substance P-Gly level was 2.4-fold higher while the substance P level was 2.1-fold lower than the corresponding peptides in control cells. These results suggest that PAM inhibition represents an effective method for suppression of substance P biosynthesis and, therefore, may have therapeutic utility in conditions associated with elevated substance P levels. Furthermore, PAM inhibition may also prove useful in decreasing other amidated peptides.The C-terminal amide is a prerequisite for full biological activity of many neuropeptides (1). These neuropeptides are typically synthesized as glycine-extended precursors and converted to the mature peptides by a family of enzymes involved in posttranslational modifications, e.g. O-glycosylation, phosphorylation, sulfation, and hydroxylation, as well as in proteolytic processing, e.g. endoproteolysis and exoproteolysis (2). The final step of posttranslational processing is catalyzed by an enzyme originally identified as peptidylglycine ␣-amidating monooxygenase (PAM) 1 (3-5). PAM is localized in secretory granules and requires copper, ascorbate, and molecular oxygen for activity (2, 6, 7). Recent studies have shown that PAM is actually a bifunctional enzyme that contains two distinct enzymatic activities and catalyzes the C-terminal amidation in a sequential manner. The first enzyme, peptidylglycine ␣-hydroxylating monooxygenase (PHM) (EC 1.14.17.3), requires the cofactors for activity and catalyzes the peptidylglycine ␣-hydroxylation reaction while the second enzyme, peptidyl ␣-hydroxyglycine ␣-amidating lyase (PAL) (EC 4.3.2.5), converts the intermediate into an ␣-amidated peptide and glyoxylate (8 -11). Among the glycine-extended neuropeptides examined, glycine-extended substance P (substance P-Gly) has been demonstrated to possess the highest affinity ...

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The Enzymatic Formation of Novel Bile Acid Primary Amides

King

Barnes

Glufke

et al. 2000

Archives of Biochemistry and Biophysics

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Inhibition of Peptidylglycine .alpha.-Amidating Monooxygenase by N-Substituted Homocysteine Analogs

Cited by 24 publications

References 18 publications

Pyruvate-extended Amino Acid Derivatives as Highly Potent Inhibitors of Carboxyl-terminal Peptide Amidation

Pyruvate-extended Amino Acid Derivatives as Highly Potent Inhibitors of Carboxyl-terminal Peptide Amidation

Suppression of Substance P Biosynthesis in Sensory Neurons of Dorsal Root Ganglion by Prodrug Esters of Potent Peptidylglycine α-Amidating Monooxygenase Inhibitors

The Enzymatic Formation of Novel Bile Acid Primary Amides

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