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/proteins are susceptible to C-terminal degredation by carboxypeptidase. An analysis of whether E. coli can be modified to perform amidation will be followed by a summary of the research in the conclusion.
Review and Discussion
What is a Peptide? A peptide is any organic substance of which the 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 that involve 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 the 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).
Further, 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 being largely academic; some authorities have adopted, as an upper limit on the molecular weight of a polypeptide, 10,000 (that of a peptide that is composed of about 100 amino acids) (Conley, Schwartz & Desforges, 2004).
The synthesis of peptides is of enormous interest to researchers because an important natural polypeptide has confirmed the structure assigned to it. One of the most important synthetic methods developed in the past was that of Bergmann and Zervas. This approach is based upon the fact that carbobenzoxy (C6H5CH2OCO-) derivatives of amino acids may be split by catalytic hydrogenation. Among other recent methods, there is the conversion of amino acids into mixed anhydrides with carbonic acid; these latter compounds react with an amino group to form a peptide bond. Likewise, carbobenzoxy-amino acid anhydrides react readily with other amino acids (Florkin, 1960).
Figure 1. Peptide or Amide Synthesis [Source: Ophart, 2003].
What is Amidation of a Peptide? An amidase is any enzyme that hydrolyzes acid amides, generally with the liberation of ammonia taking place (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 that is 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).
Importance of Amidation of Peptides. 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 have in amidated peptide production (Merkler, 1994).
These processes are assuming increasing commercial and medical significance in the 21st century as well (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 wouldl average 15%-20%/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 UCB-Bioproducts. Scott writes, "The range of medicinal uses for peptides is almost unlimited" (Scott, 2002, p. 27). The company made the comments at a meeting that was hosted by the Taiwan government's Biotechnology & Pharmaceutical Industries Program Office (BPIPO) in Taipei.
According to the report, UCB-Bioproducts claims to be the largest company in the world specifically focused on the manufacture of peptide-based APIs, Bille says. UCB-Bioproducts has been concentrating on the contract API business, and is employing its expertise in synthetic peptide processes to assist its clients, who are major pharmaceutical and biotech companies -- through regulatory procedures prior to a peptide therapy's market introduction, the company says (Scott, 2002). To date, the company has no plans to establish manufacturing facilities elsewhere; however, it is interested in increasing sales to the 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). Finally, 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 three years or so, 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. The company provides the life-science industry peptides based on the use of three following 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 says.
Lonza also has extensive custom experience in the synthesis of peptides using so called 'hybridal 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 such as by using 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 the animated peptide products and is targeting its services toward both pharmaceutical and biopharmaceutical companies that require peptides for a large 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 commercial production of synthetic peptides. The new echnology combines the best attributes of the two main peptides production technologies, solid-phase and solution-phase production, and at the commercial scale could be "significantly cheaper and offer manufacturing flexibility," Diosynth says. Solid-phase peptide production typically takes less…