Amino Acids, Peptides, and Proteins Help (page 2)
This study guide deals with the very important α-amino acids, the building blocks of the proteins that are necessary for the function and structure of living cells. Enzymes, the highly specific biochemical catalysts, are proteins.
Amino acids are zwitterions: they have both + and - charges in them at neutral pH. The natural amino acids have different side-chains (R groups), but the same α-aminocarboxylic acid backbone. With the exception of glycine (R=H), the natural amino acids are chiral, with the S configuration at the α-carbon.
The 20 most common naturally occurring amino acids are listed below. They all are primary amines, except for proline which is a 2° amine. They are grouped into different catagories, depending on the chemical nature of the side-chain group. Each amino acid has a 3-letter abbreviation.
Chemical Properties of Amino Acids
Acid-Base (Amphoteric) Properties. The pH at which [anion] = [cation] is called the isoelectric point. At this pH, there is no net charge on the molecule. The different amino acids have different isoelectric points, depending on whether the side-chain is acid, basic, or neutral.
Peptides are polyamides composed of the different amino acids. The amide bond between two amino acid residues is often called a peptide bond. Peptides can range from simple 2-amino acid residue compounds up to long chains of 50 to 100 residues, at which point they approach the size of proteins.
The artificial sweetener aspartame is a synthetic dipeptide, the methyl ester of aspartylphenylalanine, or AspPheOCH3.
It is possible to completely hydrolyze a peptide to its component amino acids by heating in aqueous acid. While this can, in principal, provide the composition of the peptide, it does not reveal the order in which the amino acids occur in the chain. This information (the sequence) is determined by other methods.
Sequencing. By cutting off one terminal amino acid at a time from either the free amino end (N-terminus) or the free COOH end (C-terminus), sequencing techniques give the linkage order (primary structure) of the amino acids in the peptide.
The N-terminal residue can be identified with either the Sanger method or the Edman method. In the Sanger method, the free NH2 group of the N-terminal residue reacts with 2,4-dinitrofluorobenzene (DNFB) in an unusual example of nucleophilic aromatic substitution. After complete hydrolysis of the peptide, the DNFB-labelled amino acid can be isolated and identified.
The Edman degradation is similar, although phenylisothiocyanate is used to label the N-terminal residue, and a phenylthiohydantoin is isolated. The Edman degradation can be repeated to identify each residue in turn.
Carboxypeptidase, an enzyme, selectively cleaves the C-terminal amino acid from a peptide chain, and so can be used to identify each residue in turn.
The synthesis of peptides from amino acids requires the use of protecting groups to prevent random coupling of amino acids and polymerization. These groups can be installed to render a functional group unreactive, then removed to liberate the reactive functional group.
Blocking the N-Terminal Amino Group. This reaction is achieved by attaching a group (B) to N of NH2. After the desired peptide is constructed, the blocking group is removed without destroying the peptide linkages. A common group is the t-butoxycarbonyl, or "Boc" group:
Activating the COOH. The –COOH is not reactive enough on its own to form an amide. It is first activated to form a peptide bond with an N-terminal NH2 by reacting the N-blocked amino acid with ethyl chloroformate (EtOC(O)Cl). A mixed anhydride of an alkyl carbonic acid is formed. This technique minimizes racemization of the chiral α-carbon
The COOH groups can also be activated in the presence of free N-terminal amino groups with N,N-dicyclohexylcarbodimide (DCC).
Coupling. A CO2H-activated, Boc protected amino acid can be coupled with an amino acid with a free NH2 group to make a dipeptide.
The synthesis can be continued, or the protecting groups can be hydrolyzed off with aqueous acid to liberate the free peptide.
Merrifield Solid-Phase Synthesis. A major advance in peptide synthesis is the solid-phase method. Beads of solid chloromethylated polystyrene [chloromethyl groups (–CH2Cl) para to about every hundredth phenyl group] are used in an automated process. The sequence of steps is: (1) a C-terminal Boc-protected amino acid is attached by substitution for the Cl of the –CH2Cl group; (2) the Boc group is removed; (3) a Boc-protected amino acid is added and the new peptide bond is formed with the aid of DCC. Steps (2) and (3) are repeated as many times as is necessary to make the desired peptide. In the final step, the completed peptide is detached by hydrolysis of the ester bond that holds the peptide to the solid surface. The average yield for each step exceeds 99%.
Proteins are polypeptides with molecular weights from ca. 10,000 up to several million, and are a major constituent of living cells. Simple proteins are hydrolyzed to amino acids. Conjugated proteins are composed of amino acids and nonpeptide substances known as prosthetic groups. These prosthetic groups include nucleic acids of nucleoproteins, carbohydrates of glycoproteins, pigments (such as hemin and chlorophyll) of chromoproteins, and fats or lipids of lipoproteins.
Amphoteric Properties. Isoelectric Points and Electrophoresis. Proteins have different isoelectric points, and in an electrochemical cell they migrate to one of the electrodes (depending on their charge, size, and shape) at different speeds. This difference in behavior is used in electrophoresis for the separation and analysis of protein mixtures.
The primary structure of a protein consists of the sequence of the constituent amino acids. The secondary structure arises from different conformations of the protein chains; these conformations are best determined by X-ray analysis. There are three types.
- The α-helix is a mainly right-handed coiled arrangement maintained by H-bonds between an N–H and O=C that are four peptide bonds apart.
- The pleated sheet has chains lying side by side and linked through N–H – – – – O=C H bonds. The α C's rotate slightly out of the plane of the merger (to minimize repulsions between their bulky R groups), which gives rise to the "pleats."
- Random structures have no repeating geometric pattern. However, there are structural constraints on the randomness leading to a constrained random orientation.
Tertiary structure is determined by any folding of the chains. There are two types.
- Fibrous proteins are water-insoluble, elongated, threadlike helixes (occasionally pleated sheets) made up of chains which are bundled together intermolecularly through N–H – – – – O=C H-bonding. They include fibroin (found in silk), keratin (in hair, skin, feathers, etc.), and myosin (in muscle tissue).
- Globular proteins, or globulins, are folded into compact spheroid shapes such that hydrophilic R groups point outward toward the water solvent and the hydrophobic (lipophilic) R groups turn inward. As a result, globulins can dissolve in or easily emulsify with water. The shape is maintained by intramolecular H bonding. The secondary structure is a combination of random (always present), helical, and pleated structures. Globulins include all enzymes, antibodies, albumin of eggs, hemoglobin, and many hormones such an insulin.
Quaternary structure exists when two or more polypeptide chains are linked only by weak forces of attraction among R groups at the surface of the chains. Such proteins are called oligomers (dimers, trimers, and so on).
Denaturation. Heat, strong acids or bases, ethanol, or heavy-metal ions irreversibly alter the secondary structure of proteins (see below). This process, known as denaturation, is exemplified by the heat-induced coagulation and hardening of egg white (albumin). Denaturation destroys the physiological activity of proteins.
Practice problems for these concepts can be found at: Amino Acids, Peptides, and Proteins Practice Problems
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