Peptide Amidation: PAM Enzyme, PHM, PAL, and E. coli

Introduction to Peptides and Amidation

Modern biotechnology has experienced dramatic leaps in the body of knowledge concerning molecular processes in peptides and how they work. Many of these processes rely on amidation of peptides to achieve increasingly important medical and commercial applications. Peptides are created when two or more amino acids are covalently joined by peptide bonds, a process termed post-translational modification. One increasingly valuable application of post-translational modification is amidation. This paper provides an overview of peptides and their role in biological processes, how amidation of peptides works and its importance, and a description of the two functional domains of the PAM enzyme (PHM and PAL) and the roles they play in amidation. An assessment of whether amidation prevents C-terminal degradation is followed by a discussion of which peptides and proteins are susceptible to C-terminal degradation by carboxypeptidase. An analysis of whether E. coli can be modified to perform amidation concludes the review.

A peptide is any organic substance whose molecules are structurally similar to those of proteins, but of smaller size (Conley, Schwartz & Desforges, 2004). The class of peptides includes many hormones, antibiotics, and other compounds that are active in the metabolic functions of living organisms. Peptide molecules are comprised of two or more amino acids joined through amide formation involving the carboxyl group of each amino acid and the amino group of the next (Audesirk & Audesirk, 1993). The chemical bond between the carbon and nitrogen atoms of each amide group is known as a peptide bond; some or all of the peptide bonds, which connect the consecutive triplets of atoms in the chain regarded as the backbone of the molecule, can be broken by partial or complete hydrolysis of the compound (Conley, Schwartz & Desforges, 2004). According to Florkin (1960), the peptide bond is an amide linkage resulting from the reaction of a carboxyl group with an amino group, together with the elimination of water. Peptides are the result of joining two or more amino acids by peptide linkage, thereby producing smaller peptides and finally the individual amino acids; this reaction is commonly used in studies of the composition and structure of peptides and proteins (Conley, Schwartz & Desforges, 2004).

There are currently methods to determine peptide binding to some HLA class II-DR and -DQ molecules; some of these methods measure the relative strength of the peptide-HLA interaction using isolated class II molecules and purified peptides. Other methods are used to predict peptide binding to HLA using computer algorithms (Harding, Mucha, Power & Stickler, 2003). The number of amino-acid molecules present in a peptide is indicated by a prefix: a dipeptide contains two amino acids; an octapeptide, eight; an oligopeptide, a few; a polypeptide, many (Conley, Schwartz & Desforges, 2004). The distinction between a polypeptide and a protein is imprecise and is regarded as largely academic; some authorities have adopted, as an upper limit on the molecular weight of a polypeptide, 10,000 (that of a peptide composed of about 100 amino acids) (Conley, Schwartz & Desforges, 2004).

The synthesis of peptides is of enormous interest to researchers because important natural polypeptides have confirmed the structures assigned to them. One of the most important synthetic methods developed in the past was that of Bergmann and Zervas. This approach is based on the fact that carbobenzoxy (C₆H₅CH₂OCO-) derivatives of amino acids may be split by catalytic hydrogenation. Among other recent methods is the conversion of amino acids into mixed anhydrides with carbonic acid; these compounds react with an amino group to form a peptide bond. Likewise, carbobenzoxy-amino acid anhydrides react readily with other amino acids (Florkin, 1960).

An amidase is any enzyme that hydrolyzes acid amides, generally with the liberation of ammonia (Florkin, 1960). The amidation of peptides concerns the posttranslational conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides (Peptide amidation, 2004). Amidation takes place in over half of all peptide hormones to produce bioactive peptides. This is a two-step function catalyzed by a peptidyl-glycine alpha-hydroxylating monooxygenase and a peptidyl-alpha-hydroxyglycine alpha-amidating lyase; in some organisms, this process is catalyzed by two separate enzymes, whereas in higher organisms, one polypeptide catalyzes both reactions (Peptide amidation, 2004).

According to Merkler (1994), peptidylglycine alpha-amidating enzyme (alpha-AE) can be used in an in vitro reaction to convert C-terminal glycine-extended peptides to peptide hormones with a C-terminal amino acid amide. Structure-activity data for 45 bioactive peptides show that the C-terminal amide is required for the full biological activity of most amidated peptide hormones. These findings emphasize the role alpha-AE can play in amidated peptide production (Merkler, 1994).

Importance of Peptide Amidation: Medical and Commercial Applications

These processes are assuming increasing commercial and medical significance in the 21st century (Scott, 2002). For instance, according to a report from Chemical Week, UCB-Bioproducts (Braine-l'Alleud, Belgium) predicted that sales growth in the peptide-based active pharmaceutical ingredients (API) market would average 15–20% per year, fully double that of the overall API market growth. UCB also reported that the application of peptide-based therapies will become broader with the emergence of new drug-delivery systems, including skin patches, inhalers, and sprays that offer a promising alternative to the syringes commonly used today (Scott, 2002).

Peptide-based therapies such as insulin are easily degraded by digestive enzymes and are usually injected rather than administered orally. Emerging applications for peptide-based therapies will also add to demand growth, UCB says. "There are new applications in cardiovascular medicine, for cancer treatment, for metabolic disorders, as antibacterial and antiviral agents," says Vincent Bille, peptides business unit manager at UCB-Bioproducts. As Scott writes, "The range of medicinal uses for peptides is almost unlimited" (Scott, 2002, p. 27). The company made these comments at a meeting hosted by the Taiwan government's Biotechnology & Pharmaceutical Industries Program Office (BPIPO) in Taipei.

UCB-Bioproducts claims to be the largest company in the world specifically focused on the manufacture of peptide-based APIs. UCB-Bioproducts has been concentrating on the contract API business and is employing its expertise in synthetic peptide processes to assist its clients — major pharmaceutical and biotech companies — through regulatory procedures prior to a peptide therapy's market introduction (Scott, 2002). The company has no current plans to establish manufacturing facilities elsewhere; however, it is interested in increasing sales to the Asia-Pacific region, says Roy Kosasih, Asia/Pacific sales and marketing manager: "We believe we can help small companies in many ways, particularly when such companies have produced new life science technologies but don't know how to bring them to market" (Scott, 2002, p. 27). UCB-Bioproducts has peptide manufacturing facilities at Braine-l'Alleud and an application laboratory in Smyrna, Georgia. The company's sales for 2001 were $39 million. "Marketing efforts have attracted new projects, which will ensure the growth of this activity in the future," the firm reported (Scott, 2002, p. 27).

Another company, Lonza, based in Switzerland, has also entered the mass production of peptides. Over the past several years, the company has gained a considerable reputation in the industry and produces hundreds of kilograms of peptides from its facilities at Visp, Switzerland. The company has further investment programs planned for a substantial capacity increase in cGMP production. Lonza provides the life-science industry with peptides based on three technologies: solid-phase; liquid solution-phase; and recombinant technology in conjunction with Lonza's Biotechnology division. A combination of all three types of technology is another option Lonza offers.

Lonza also has extensive custom experience in the synthesis of peptides using what it calls "hybrid technology," according to Chemical Week. This technology involves the production of fragments on solid support and the subsequent condensation of those fragments in liquid-phase, followed by reverse high-pressure liquid chromatography (HPLC) purification. This approach has been shown to be more efficient and cost-effective. Lonza employs a variety of small and large-scale solid-phase peptide synthesizers (SPPS) with capacities ranging from laboratory scale to production in 250-liter to 850-liter vessels. Lonza also has the necessary technology platform for subsequent modification of the peptide intermediate, including amidation technology (Lonza extends peptides and oligo-nucleotides capabilities, 2003).

Lonza is able to provide a one-stop-shop strategy for the entire life cycle of amidated peptide products and is targeting its services toward both pharmaceutical and biopharmaceutical companies that require peptides for a wide variety of therapeutic applications, including HIV, cancer, and diabetes (Lonza extends peptides and oligo-nucleotides capabilities, 2003).

Finally, Diosynth, Akzo Nobel's active pharmaceutical ingredients subsidiary, reports that it has developed a novel high-speed process for the commercial production of synthetic peptides. The new technology combines the best attributes of the two main peptide production technologies — solid-phase and solution-phase production — and at commercial scale could be "significantly cheaper and offer manufacturing flexibility," Diosynth says. Solid-phase peptide production typically takes less time than solution-phase processes, but is less suited to commercial-scale production because it requires significant modification when scaling up to maintain the integrity of the reaction. The commercial development and production of a peptide using a solution-phase process can take up to two years, due in part to the requirement that after each step the peptide must be isolated from the solution. The new Diosynth 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, with a separate reaction step 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 amino 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 already used it 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 ranging from 100 liters to 10 cubic meters. The increased flexibility and acceleration of peptide production will significantly improve Diosynth's capacity. 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).

Peptidylglycine alpha-amidating monooxygenase (PAM) is a multifunctional protein found in secretory granules (Peptide amidation, 2004). The PAM protein contains two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides:

How Amidation Occurs: The PAM Enzyme and Its Two Functional Domains

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 note, "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, PAM; the PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), generates the 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 as well (Prigge et al., 2000).

Conclusion

The research showed that peptides are formed when two or more amino acids are covalently joined by peptide bonds, a process termed post-translational modification. In vertebrates, amidated peptides provide a number of useful functions, serving as hormones, neurotransmitters, and paracrine agents. One increasingly valuable use of post-translational modification is amidation. Amidation is being used for a wide variety of innovative medical and commercial applications.