Enzymatic peptidyl .alpha.-amidation proceeds through formation of an .alpha.-hydroxyglycine intermediate

Young, Stanley D.; Tamburini, Paul P.

doi:10.1021/ja00187a088

Cited by 113 publications

(58 citation statements)

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“…Our earlier studies on mammalian pituitary amidation established that in the initial step, the p r o s hydrogen of the glycine residue is removed (Ramer et al, 1988(Ramer et al, ,1989 and that aerobic oxygen gas contributes the oxygen atom of the hydroxy group (Za-briskie et al, 1991). Subsequent work on other mammalian PHMs has confirmed the aerobic origin of the hydroxy group (Merkler et al, 1992a;Noguchi et al, 1992) and has demonstrated that in bovine pituitary the process occurs with retention of configuration to form an (S)-a-hydroxy-Gly peptide (Ping et al, 1992), which has the correct stereochemistry to be accepted for cleavage to peptide amide and glyoxylate by PAL (Young and Tamburini, 1989;Ping et al, 1992). Both the insect and bovine pituitary PHMs produce an (S)-a-hydroxy-Gly peptide (i.e., 7a) and will not accept peptides bearing C-terminal L-amino acids in place of glycine (see below) but will transform certain substrate analogues ending with a D-amino acid.…”

Section: Stereochemical Aspects and Inhibition Of Bee Phm By Substratmentioning

confidence: 95%

Peptide amidation in an invertebrate: Purification, characterization, and inhibition of peptidylglycine α‐hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

Zabriskie

Klinge

Szymanski

et al. 1994

Arch Insect Biochem Physiol

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Peptidylglycine alpha-hydroxylating monooxygenase (PHM), an enzyme involved in formation of neuropeptides with a C-terminal amide functionality in mammals and amphibians, was isolated from the head of an invertebrate, the honeybee, Apis mellifera, and purified 220-fold in 1% overall yield. The bee PHM has a molecular weight of 71,000, is membrane associated but can be solubilized with a detergent (n-octyl-beta-D-glucopyranoside), and cross-reacts with rabbit antibodies generated toward bacterially expressed rat PHM. In the presence of copper, oxygen, and ascorbic acid, the enzyme hydroxylates model tripeptides such as dansyl-L-Phe-L-Phe-Gly on the methylene carbon of the glycine residue with retention of configuration. Using this tripeptide as substrate, the Km is 1.7 microM and the Vmax is 2.3 nmol.micrograms-1.h-1. Treatment of the insect PHM with D-Phe-L-Phe-D-vinylglycine, a substrate analogue and mechanism-based inactivator of PHM from pig pituitary, results in irreversible loss of activity. The diastereomeric analogue, D-Phe-L-Phe-L-vinylglycine, is only a competitive inhibitor (IC50 = 320 microM).

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Section: Stereochemical Aspects and Inhibition Of Bee Phm By Substratmentioning

confidence: 95%

Peptide amidation in an invertebrate: Purification, characterization, and inhibition of peptidylglycine α‐hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

Zabriskie

Klinge

Szymanski

et al. 1994

Arch Insect Biochem Physiol

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“…1) is a bifunctional enzyme catalyzing the two-step conversion of peptides with a COOHterminal glycine residue into a-amidated peptides; peptide a-amidation is a late step in peptide processing, 0888-8809/91/0187-0193S03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society beginning in the frans-Golgi network, but occurring primarily in secretory granules (1,(6)(7)(8)(9)(10)(11). The first (monooxygenase) step of the reaction depends on copper ions and oxygen and consumes reduced ascorbate; the second (lyase) step results in cleavage of the peptidyla-hydroxyglycine intermediate to yield the a-amidated peptide and glyoxylate (8,(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Manipulation of Neuropeptide Biosynthesis through the Expression of Antisense RNA for Peptidylglycine α-Amidating Monooxygenase

Mains

Bloomquist

Eipper

1991

Molecular Endocrinology

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Stable cell lines with significantly elevated or diminished levels of a key neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), were generated by transfection of a mouse pituitary cell line with expression vectors containing PAM cDNA in the sense or antisense orientation. By evaluating the ability of these cell lines to alpha-amidate endogenous neuropeptides, a rate-limiting role for PAM in neuropeptide alpha-amidation was demonstrated. Overexpression of either the full-length PAM precursor with its trans-membrane domain or a soluble protein containing only the monooxygenase domain of PAM led to increased alpha-amidation of endogenous neuropeptides. Overexpression of the full-length PAM led to an unexpected decrease in the endoproteolytic processing of endogenous prohormone; conversely, underexpression of PAM led to significantly enhanced endoproteolytic processing of endogenous prohormone. These data suggest that PAM may have additional functions in peptide processing.

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“…Amidation proceeds in two sequential steps each catalyzed by individual domains of the PAM enzyme : hydroxylation, catalyzed by the copper-containing peptidylglycine α-hydroxylating monooxygenase (PHM) and dealkylation of the carbinolamide intermediate catalyzed by the Ca/Zn containing peptidylglycine α-amidating lyase (PAL) (8, 9). Specifically, the PHM domain mediates the stereospecific hydroxylation of the terminal glycine of the peptide substrate to form a peptidyl-α-hydroxyglycine intermediate which undergoes N-C bond fission and elimination of glyoxalate to yield the α-amidated product (10–12) (Figure 1). …”

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confidence: 99%

Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

Chauhan

Hosseinzadeh

Lu³

et al. 2016

Biochemistry

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PHM is a dicopper enzyme that plays a vital role in the amidation of glycine extended pro-peptides. One of the crucial aspects of its chemistry is the transfer of two electrons from an electron-storing and transferring site (CuH) to the oxygen binding site and catalytic center (CuM) over a distance of 11 Å during one catalytic turnover event. Here we present our studies on the first electron transfer step (reductive phase) in the WT PHM as well as its variants. Stopped-flow was used to record the reduction kinetic traces using the chromophoric agent DMPD as the reductant. The reduction was found to be biphasic in the WT PHM with an initial fast phase (17.2s−1) followed by a much slower phase (0.46s−1). We were able to ascribe the fast and slow phase to the CuH and CuM-sites respectively by making use of the H242A and H107H108A mutants which only contain the CuH-site and CuM-site respectively. In the absence of substrate the redox potentials determined by cyclic voltammetry were 270 mV (CuH-site) and −15 mV (CuM-site), but binding of substrate (Ac-YVG) was found to alter both potentials so that they converged to a common value of 83 mV. Substrate binding also accelerated the slow reductive phase by ~10 fold, an effect that could be explained at least partially by the equalization of the reduction potential of the copper centers. Studies on H108A showed that the ET to the CuM-site is blocked, highlighting the role of the H108 ligand as a component of the reductive ET pathway. Strikingly, the rate of reduction of the H172A variant was unaffected despite the rate of catalysis being three orders of magnitude less than that of the WT PHM. These studies strongly indicate that the reductive phase and catalytic phase ET pathways are different and suggest a bifurcated ET pathway in PHM. We propose that H172 and Y79 form part of an alternate pathway for the catalytic phase ET while the H108 ligand along with the water molecules and substrate form the reductive phase ET pathway.

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Enzymatic peptidyl .alpha.-amidation proceeds through formation of an .alpha.-hydroxyglycine intermediate

Cited by 113 publications

References 0 publications

Peptide amidation in an invertebrate: Purification, characterization, and inhibition of peptidylglycine α‐hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

Peptide amidation in an invertebrate: Purification, characterization, and inhibition of peptidylglycine α‐hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

Manipulation of Neuropeptide Biosynthesis through the Expression of Antisense RNA for Peptidylglycine α-Amidating Monooxygenase

Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

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