Peptidylglycine α-amidating reaction: Evidence for a two-step mechanism involving a stable intermediate at neutral pH

Takahashi, Ken­ichi; Okamoto, Hiroshi; Seino, Hiroshi; Noguchi, Masato

doi:10.1016/0006-291x(90)90362-q

Cited by 78 publications

(21 citation statements)

References 13 publications

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“…0.01 pmol product/h/mg of protein). A pH optimum of 7 was determined, agreeing with previous findings of Takahashi et al [19]. The K, for the synthetic substrate was 130 nM with a standard deviation calculated by the least squares method of 20 nM.…”

Section: Pam Activity In Larval Brainsmentioning

confidence: 94%

Invertebrate neuropeptide processing: Partial characterization of peptidylglycine α‐amidating monooxygenase in the tobacco hornworm, Manduca sexta

Bernasconi¹,

Li²,

Reagan³

et al. 1992

Arch Insect Biochem Physiol

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A peptidylglycine a-amidating monooxygenase (PAM) has been characterized in the tobacco hornworm Manduca sexta. Although such enzymes have been well studied in vertebrates, their presence in invertebrates has not been reported. PAM activity was measured in several insect tissues. The highest specific activity was found in the brain. In this organ, PAM activity was high during the larval stages, but low at the end of metamorphosis. Copper, oxygen, and ascorbate were required for activity. Evidence that 2,4,5-trihydroxyphenylalanine may be involved in PAM activity is presented.

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Section: Pam Activity In Larval Brainsmentioning

confidence: 94%

Invertebrate neuropeptide processing: Partial characterization of peptidylglycine α‐amidating monooxygenase in the tobacco hornworm, Manduca sexta

Bernasconi¹,

Li²,

Reagan³

et al. 1992

Arch Insect Biochem Physiol

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“…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)(4)(5)(6)(7)(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).…”

mentioning

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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“…cDNA clones encoding the 110-kDa bifunctional integral membrane protein form of PAM have been isolated from many tissues in several species (Murthy et al 1986;Kizer et al 1986;Mizuno et al 1986;Gilligan et al 1989;Mehta et al 1988;Noguchi et al 1989;Takahashi et al 1990). Tissue-specific alternative splicing generates at least seven forms of PAM mRNA in the rat (Eipper et al 1992a).…”

Section: Introductionmentioning

confidence: 99%

Characterization and regulation of peptidylglycine α-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts

Girard¹,

Ouafik²,

Boudouresque³

1999

Cell and Tissue Research

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Peptidylglycine α-amidating monooxygenase (PAM), which catalazyes the two-step formation of bioactive α-amidated peptides from their glycine-extended precursors, has been found in H9c2 myoblasts. The expression of PAM has been evaluated in H9c2 cells. Northern blot analysis and amplification of fragments derived from rat PAM by the reverse transcription/polymerase chain reaction method has demonstrated the presence of rPAM-1, -2, -3, -3a and -3b mRNA transcripts. These forms of PAM mRNA may be generated by alternative splicing. PAM mRNA levels are increased to 160±12% of control values by treatment with dexamethasone but are unchanged during triiodothyronine incubation of the cells. PAM activity is very low, which is not comparable to the high levels found in adult atrium tissue. Western blot analysis has demonstrated 86-, 76-, and 46-kDa PAM proteins in the particulate fraction. The soluble fraction contains major PAM proteins of 110, 86, and 46 kDa. In situ hybridization studies with 35 S-labeled full length RNA antisense transcripts of rat PAM-1 cDNA have localized autoradiographic grains around the nucleus. Our data clearly demonstrate PAM expression in H9c2 rat heart cells, suggesting the ability of these cardiac cells to make bioactive α-amidated hormones and/or neuropeptides.

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Peptidylglycine α-amidating reaction: Evidence for a two-step mechanism involving a stable intermediate at neutral pH

Cited by 78 publications

References 13 publications

Invertebrate neuropeptide processing: Partial characterization of peptidylglycine α‐amidating monooxygenase in the tobacco hornworm, Manduca sexta

Invertebrate neuropeptide processing: Partial characterization of peptidylglycine α‐amidating monooxygenase in the tobacco hornworm, Manduca sexta

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

Characterization and regulation of peptidylglycine α-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts

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