Amidation of Peptides in Humans Term Paper

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[. . .] The commercial development and production of a peptide using a solution-phase process can take up to two years, due in part because after each step the peptide has to be isolated from the solution. However, the new process overcomes the drawbacks of both systems by enabling scalable peptide production over a period of weeks, according to Ralf van Dijck, the peptides product manager at Diosynth (Scott, 2003).

Pharmaceutical peptides are generated by chemically coupling amino acids; a separate reaction step is required for attaching each amino acid. In the standard solution-phase process, the peptide is isolated after the addition of each amino acid. In the Diosynth process, the peptide is anchored in the solution. The anchored peptide is retained in the reactor while uncoupled ammo acids can be eliminated. "We can go directly from the 1 gram to multikilogram scale, depending on the size of the vessel," van Dijck noted. Diosynth recently patented the technology and has used it already in order to generate a multikilogram batch of a peptide in several weeks. "It could significantly reduce the time to market for a commercial peptide," van Dijck added (Scott, 2003, p. 23). The new technology is applicable to the production of all peptides and may be applied to Diosynth's peptide facilities at Oss, the Netherlands where the company has reaction vessels that range from 100 liters, to 10 cu meters. The increased flexibility and acceleration of peptides production will significantly improve Diosynth's peptide production capacity, van Dijck says. Peptides are a key growth area for custom sales to the pharmaceutical sector: "Synthetic peptides represent an important product range with strong growth potential," according to Johan Evers, general manager at Diosynth (Scott, 2003, p. 23).

How Does Amidation Occur? Peptidylglycine alpha-amidating monooxygenase (PAM) is a multifunctional protein that is found in secretory granules (Peptide amidation, 2004).

The PAM protein contains two enzymes that act sequentially in order to catalyze the alpha-amidation of neuroendocrine peptides:

peptidylglycine + ascorbate + O2 = peptidyl (2-hydroxyglycine) + dehydroascorbate + H2O.

The resulting product is unstable and dismutates to glyoxylate and the corresponding desglycine peptide amide (Peptide amidation, 2004).

According to Prigge et al. (2000), a number of bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. "Surprisingly," they say, "the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond" (p. 1236). In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM); the PAM gene then encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation.

Peptidylglycine alpha-hydroxylating monooxygenase catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase, generates alpha-amidated peptide product and glyoxylate.

PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase, the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM (Prigge et al., 2000).

Description of the Two Functional Domains of the PAM enzyme (PHM and PAL) and the Roles they Play in Amidation. The first step of the amidation reaction is catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM), and is dependent on copper, ascorbate and molecular oxygen; peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction (Stoffers, Ouafik & Eipper, 1991). The final two steps in the biosynthesis of alpha-amidated bioactive peptides are catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) (Mueller, Husten, Mains, & Eipper, 1993).

These enzymes are derived from the bifunctional precursor protein, peptidylglycine alpha-amidating monooxygenase. Because PHM is rate-limiting in peptide amidation and is copper-dependent, we examined the consequences of in vivo treatments with the copper-chelating drug disulfiram (Antabuse) on levels of alpha-amidated peptides and expression of PHM and PAL (Mueller, Husten, Mains, & Eipper, 1993).

Decreases in two amidated peptides (alpha-melanotropin and cholecystokinin) after disulfiram treatment were extremely pronounced outside the blood-brain barrier, with moderate decreases in the central nervous system. Unexpectedly, when assayed under optimal conditions in vitro, PHM activity was increased by disulfiram treatment, whereas PAL activity was unaltered. The increase in PHM activity in pituitary and atrium occurred within a few hours after the start of disulfiram treatment and was sustained up to 2 weeks after the cessation of treatment, whereas levels of alpha-amidated peptides remained low (Mueller, Husten, Mains, & Eipper, 1993).

Northern and Western blot analyses demonstrated that disulfiram had no influence on levels of peptidylglycine alpha-amidating monooxygenase mRNA or protein. Thus, inhibition of alpha-amidation by disulfiram in vivo occurs despite an increased Vmax of PHM assayed in vitro. The increase in PHM activity may result from induction of a physiologic mechanism that normally regulates this rate-limiting enzyme (Mueller, Husten, Mains, & Eipper, 1993). Sequence analysis by these researchers showed the protein to be similar to dopamine-beta-monooxygenases (DBH), a class of ascorbate-dependent enzymes that requires copper as a cofactor and uses ascorbate as an electron donor (Mueller, Husten, Mains, & Eipper, 1993).

According to Stoffers, Ouafik and Eipper (1991), PAM and DBH share a few regions of sequence similarity, some of which contain clusters of conserved histidine residues that may be involved in copper binding. Ouafik, Stoffers, Campbell et al. (1992) note that peptidylglycine alpha-amidating monooxygenase is a multifunctional protein that contains two enzymes that also act sequentially in order to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction (Ouafik, Stoffers & Campbell, 1992).

Studies to date have demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded with a complex single copy gene that is comprised of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons that comprise the PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length (Ouafik, Stoffers & Campbell, 1992).

Researchers have also shown that the alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) rather than a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains.

The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Finally, the analysis of hybrid cell panels suggests that the human PAM gene is situated on the long arm of chromosome 5 (Ouafik, Stoffers & Campbell, 1992).

The peptidylglycine alpha-amidating enzyme catalyzes a reaction that transforms a carboxyl-terminal glycine-extended precursor into a carboxyl-terminal alpha-amidated peptide. We purified an alpha-amidating enzyme from equine serum by simplified steps including substrate affinity chromatography. With the purified enzyme, Tajima et al. discerned an intermediate of the alpha-amidating reaction by high performance liquid chromatography analysis.

The production of the intermediate required copper, oxygen, and ascorbate and increased linearly with incubation time. The structure of the intermediate was determined to be a hydroxyl derivative at the carboxyl-terminal glycine by fast atom bombardment mass spectrometry and by proton NMR. The intermediate was readily converted into an alpha-amidated product in alkaline conditions in a nonenzymic fashion. The nonenzymic conversion required no cofactor but was extremely accelerated by the addition of copper ion or at higher temperature. Our data suggest that the direct product of the alpha-amidating reaction is not an alpha-amidated peptide but a hydroxyl derivative at the alpha-carbon of the carboxyl-terminal glycine (Tajima et al., 1990).

As noted above, carboxy-terminal amidation… [END OF PREVIEW]

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APA Format

Amidation of Peptides in Humans.  (2004, July 19).  Retrieved February 20, 2019, from

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"Amidation of Peptides in Humans."  July 19, 2004.  Accessed February 20, 2019.