Amino Acids, Peptides & Proteins PDF

Amino acids & Proteins AMINO ACIDS, PEPTIDES, AND PROTEINS Overview of Protein:  Proteins are organic compounds with a high molecular weight formed of carbon, oxygen, hydrogen and nitrogen;  Also they may contain sulfur, phosphorus coloring non-protein organic groups and me...

Amino acids & Proteins AMINO ACIDS, PEPTIDES, AND PROTEINS Overview of Protein:  Proteins are organic compounds with a high molecular weight formed of carbon, oxygen, hydrogen and nitrogen;  Also they may contain sulfur, phosphorus coloring non-protein organic groups and metal ions.  They are polymers formed of subunits called amino acids linked together by peptide linkage.  Proteins are the most abundant biological macromolecules, occurring in all cells and all parts of cells.  Proteins also occur in great variety; thousands of different kinds, ranging in size from relatively small peptides to huge polymers with molecular weights in the millions, may be found in a single cell.  Moreover, proteins exhibit enormous diversity of biological function and are the most important final products of the information pathways.  Proteins are the molecular instruments through which genetic information is expressed.  Relatively simple monomeric subunits provide the key to the structure of the thousands of different proteins.  All proteins, whether from the most ancient lines of bacteria or from the most complex forms of life, are constructed from the same ubiquitous set of 20 amino acids, covalently linked in characteristic linear sequences.  Because each of these amino acids has a side chain with distinctive chemical properties, this group of 20 precursor molecules may be regarded as the alphabet in which the language of protein structure is written.  What is most remarkable is that cells can produce proteins with strikingly different properties and activities by joining the same 20 amino acids in many different combinations and sequences.  From these building blocks different organisms can make such widely diverse products as enzymes, hormones, antibodies, transporters, muscle fibers, the lens protein of the eye, feathers, spider webs, rhinoceros horn, milk proteins, antibiotics, mushroom poisons, and myriad other substances having distinct biological activities.  Among these protein products, the enzymes are the most varied and specialized. Virtually all cellular reactions are catalyzed by enzymes. Biological importance of proteins: 1.Nutritional role: Provide the body with essential amino acids, nitrogen and sulfur. 2.Catalytic role: All enzymes are proteins in nature. 3.Hormonal role: Most of hormones and all receptors are protein in nature. 4.Defensive role: The antibodies (immunoglobulins) that play an important role in the body‘s defensive mechanisms are proteins in nature. 5.Plasma proteins are responsible for most effective osmotic pressure of the blood. Blood clotting factors are also plasma proteins.  This osmotic pressure plays a central role in many processes, e.g., urine formation. 6.Transport role: Proteins carry lipids in the blood forming lipoprotein complexes.  Proteins also carry, hormones, e.g., thyroid hormones and minerals, e.g., calcium, iron and copper. Hemoglobin (a chromo-protein) carries O2 from the lung to tissues is a protein. 7.Structural role: Proteins are the main structural component in bone, muscles, cytoskeleton and cell membrane. By Henok. G 1  Amino acids & Proteins 8.Control of gene expression: Most factors required for DNA replication, transcription and mRNA translation are protein in nature. Amino Acids  Amino acids are organic acids that contain NH2 group. They are the structural units of proteins and are obtained from them by hydrolysis.  Twenty different amino acids are commonly found in proteins. The first to be discovered was asparagine, in 1806. The last of the 20 to be found, threonine, was not identified until 1938.  All the amino acids have trivial or common names, in some cases derived from the source from which they were first isolated;  Asparagine was first found in asparagus, and glutamate in wheat gluten; tyrosine was first isolated from cheese (its name is derived from the Greek tyros, ―cheese‖); and glycine (Greek glykos, ―sweet‖) was so named because of its sweet taste.  Functions of amino acids: Apart from being the constituents of proteins, amino acids serve a variety of functions. The non-protein amino acids along with polymerizable amino acid in their free form carry vital biochemical functions, e.g. they are urea cycle intermediates, and precursors for and precursors for neurotransmitters, polyamines, thyroid and adrenal medullary hormone synthesis (See Box). Functions of amino acids They are polymerized to form proteins. They stabilize the 3-D structure of proteins by forming hydrogen and disulfide bonds. Presence of specific amino acids at the active site of enzymes is vital for their catalytic activity Some amino acids (glucogenic) can be converted to carbohydrates. Cysteine and methionine are sources of sulfur in the body. The carbon skeleton and nitrogen of amino acids is used for the synthesis of purine and pyrimidine bases for nucleotides and nucleic acids. Glycine and methionine help in the detoxification mechianisms. Methionine can act as a methyl group donor in methylation reactions. Certain amino acids give rise to biologically improtant derivatives: oGycine is a precursor for ‘heme’ of hemoglobin and creatine that acts as the mediator of energy in muscles. oTyrosine is the precursor for a number of hormones (Thyroxine, triiodothyronine, epinephrine and nor-epinephrine) and skin pigment melanin. oTryptophan can give rise to vitamin niacin and reduce its dietary requirement and gives rise to the neurotransmitter, Serotonin. oHistidine can be converted to the mediator of allergic reactions i.e. histamine. By Henok. G 2  Amino acids & Proteins Amino Acids Share Common Structural Features:  All 20 of the common amino acids are -amino acids. They have a carboxyl group and an amino group bonded to the same carbon atom (the  carbon) (Fig. AA-1).  They differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge, and which influence the solubility of the amino acids in water.  In addition to these 20 amino acids there are many less common ones. Some are residues modified after a protein has been synthesized; others are amino acids present in living organisms but not as constituents of proteins. FIGURE AA-1 General structure of an amino acid. This structure is common to all but one of the -amino acids. (Proline, a cyclic amino acid, is the exception.) The R group or side chain (red) attached to the  carbon (blue) is different in each amino acid.  The common amino acids of proteins have been assigned three-letter abbreviations and one-letter symbols (Table–1), which are used as shorthand to indicate the composition and sequence of amino acids polymerized in proteins.  Two conventions are used to identify the carbons in an amino acid—a practice that can be confusing. The additional carbons in an R group are commonly designated β, γ, δ, ε, and so forth, proceeding out from the  carbon.  For most other organic molecules, carbon atoms are simply numbered from one end, giving highest priority (C-1) to the carbon with the substituent containing the atom of highest atomic number.  Within this latter convention, the carboxyl carbon of an amino acid would be C-1 and the  carbon would be C-2.  In some cases, such as amino acids with heterocyclic R groups, the Greek lettering system is ambiguous and the numbering convention is therefore used.  For all the common amino acids except glycine, the  carbon is bonded to four different groups: a carboxyl group, an amino group, an R group, and a hydrogen atom (Fig. AA-1; in glycine, the R group is another hydrogen atom).  The -carbon atom is thus a chiral center.  Because of the tetrahedral arrangement of the bonding orbitals around the -carbon atom, the four different groups can occupy two unique spatial arrangements, and thus amino acids have two possible stereoisomers. By Henok. G 3  Amino acids & Proteins  Since they are nonsuperimposable mirror images of each other (Fig. AA-2), the two forms represent a class of stereoisomers called enantiomers.  All molecules with a chiral center are also optically active—that is, they rotate plane-polarized light. FIGURE AA-2 Stereoisomerism in -amino acids. The two stereoisomers of alanine, L- and D-alanine, are nonsuperimposable mirror images of each other (enantiomers). Special nomenclature has been developed to specify the absolute configuration of the four substituents of asymmetric carbon atoms. The absolute configurations of simple sugars and amino acids are specified by the D, L system (Fig. AA-3), based on the absolute configuration of the three-carbon sugar glyceraldehyde, a convention proposed by Emil Fischer in 1891.  For all chiral compounds, stereoisomers having a configuration related to that of L- glyceraldehyde are designated L, and stereoisomers related to D-glyceraldehyde are designated D.  The functional groups of L-alanine are matched with those of L-glyceraldehyde by aligning those that can be interconverted by simple, one-step chemical reactions.  Thus the carboxyl group of L-alanine occupies the same position about the chiral carbon as does the aldehyde group of L-glyceraldehyde, because an aldehyde is readily converted to a carboxyl group via a one-step oxidation. Historically, the similar l and d designations were used for levorotatory (rotating light to the left) and dextrorotatory (rotating light to the right).  However, not all L-amino acids are levorotatory, and the convention shown in Figure AA-3 was needed to avoid potential ambiguities about absolute cofiguration.  By Fischer‘s convention, L and D refer only to the absolute configuration of the four substituents around the chiral carbon, not to optical properties of the molecule. FIGURE AA-3 Steric relationship of the stereoisomers of alanine to the absolute configuration of L- and D-glyceraldehyde. By Henok. G 4  Amino acids & Proteins The Amino Acid Residues in Proteins Are L-Stereoisomers: Nearly all biological compounds with a chiral center occur naturally in only one stereoisomeric form, either D or L.  The amino acid residues in protein molecules are exclusively L stereoisomers.  D-Amino acid residues have been found only in a few, generally small peptides, including some peptides of bacterial cell walls and certain peptide antibiotics. Cells are able to specifically synthesize the L isomers of amino acids because the active sites of enzymes are asymmetric; causing the reactions they catalyze to be stereospecific. Classification of Amino Acids Amino acids can be classified by one of three methods: Chemical Classification*** Biological Classification Metabolic Classification 1.Chemical Classification:-Amino Acids Can Be Classified by R Group Knowledge of the chemical properties of the common amino acids is central to an understanding of biochemistry. The topic can be simplified by grouping the amino acids into five main classes based on the properties of their R groups (Table–1), in particular, their polarity (i.e. tendency to interact with water) and charge at biological pH (near pH 7.0). The polarity of the R groups varies widely, from nonpolar and hydrophobic (water- insoluble) to highly polar and hydrophilic (water-soluble). These five classes are: Nonpolar, aliphatic R groups; Aromatic R groups; Polar, uncharged R groups; positively charged R groups; negatively charged R groups. i.Nonpolar, Aliphatic R Groups: The R groups in this class of amino acids are nonpolar and hydrophobic. The side chains of alanine, valine, leucine, and isoleucine tend to cluster together within proteins, stabilizing protein structure by means of hydrophobic interactions. Glycine has the simplest structure. Although it is formally nonpolar, its very small side chain makes no real contribution to hydrophobic interactions. Methionine, one of the two sulfur-containing amino acids, has a nonpolar thioether group in its side chain. Proline has an aliphatic side chain with a distinctive cyclic structure. The secondary amino (imino) group of proline residues is held in a rigid conformation that reduces the structural flexibility of polypeptide regions containing proline. By Henok. G 5  Amino acids & Proteins ii.Aromatic R Groups: Phenylalanine, tyrosine, and tryptophan, with their aromatic side chains, are relatively nonpolar (hydrophobic). All can participate in hydrophobic interactions. The hydroxyl group of tyrosine can form hydrogen bonds, and it is an important functional group in some enzymes. Tyrosine and tryptophan are significantly more polar than phenylalanine, because of the tyrosine hydroxyl group and the nitrogen of the tryptophan indole ring. Tryptophan and tyrosine, and to a much lesser extent phenylalanine, absorb ultraviolet light. This accounts for the characteristic strong absorbance of light by most proteins at a wavelength of 280 nm, a property exploited by researchers in the characterization of proteins. iii.Polar, Uncharged R Groups: The R groups of these amino acids are more soluble in water, or more hydrophilic, than those of the nonpolar amino acids, because they contain functional groups that form hydrogen bonds with water. This class of amino acids includes serine, threonine, cysteine, asparagine, and glutamine. By Henok. G 6  Amino acids & Proteins The polarity of serine and threonine is contributed by their hydroxyl groups; that of cysteine by its sulfhydryl group; and that of asparagine and glutamine by their amide groups. Asparagine and glutamine are the amides of two other amino acids also found in proteins, aspartate and glutamate, respectively, to which asparagine and glutamine are easily hydrolyzed by acid or base. Cysteine is readily oxidized to form a covalently linked dimeric amino acid called cystine, in which two cysteine molecules or residues are joined by a disulfide bond (Fig. AA-4). The disulfide-linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming covalent links between parts of a protein molecule or between two different polypeptide chains. Disulfide bonds between Cys residues stabilize the structures of many proteins. FIGURE AA-4 Reversible formation of a disulfide bond by the oxidation of two molecules of cysteine. By Henok. G 7  Amino acids & Proteins iv.Positively Charged (Basic) R Groups: The most hydrophilic R groups are those that are either positively or negatively charged. The amino acids in which the R groups have significant positive charge at pH 7.0 are:  Lysine, which has a second primary amino group at the ε position on its aliphatic chain;  Arginine, which has a positively charged guanidino group; and  Histidine, which has an imidazole group. Histidine is the only common amino acid having an ionizable side chain with a pKa near neutrality. In many enzyme-catalyzed reactions, a His residue facilitates the reaction by serving as a proton donor/acceptor. v.Negatively Charged (Acidic) R Groups: The two amino acids having R groups with a net negative charge at pH 7.0 are aspartate and glutamate, each of which has a second carboxyl group. By Henok. G 8  Amino acids & Proteins 2.Biological or Nutritional classification: Based upon whether the amino acids can be synthesized in human body or not: Some amino acids can not be synthesized inside the body. If these amino acids are not taken in diet they will affect the growth and the health. Thus, amino acids may be classified into: Indispensable or essential amino acids: Not synthesized in the body and must be supplied in the diet. Dispensable or non-essential amino acids: Can be synthesized in the body and hence is not essential to be present in diet. i.Essential amino acids: (Indispensable amino acids) These are amino acids that can not be synthesized in the human body and should be taken in the diet, otherwise their deficiency will lead to a nutrition deficiency disease that affect both growth and health. Arginine and histidine are semi-essential, i.e., they are mainly required in growing children, pregnant and lactating women and convalescent patients. The main sources for these amino acids are animal proteins (milk, egg, meat, liver, fish, and chicken) and a few plant proteins (bean and lintels). They are as follows (VITTAL LyMPH) or (PVT TIM HALL): Valine Isoleucine Threonine Tryptophan Arginine Leucine Lysine Methionine Phenylalanine Histidine ii.Non essential amino acids: (Dispensable amino acids) The rest of amino acids can be synthesized inside the human body and their deficiency in diet does not affect the growth or the health. 3.Metabolic Classification: Based upon the fate of amino acid inside the body: Glucogenic amino acids that can be converted to glucose. Ketogenic amino acids that can be converted to ketone bodies. Mixed function amino acids, i.e., can be converted to both glucose and ketone bodies. Ketogenic Ketogenic & glucogenic Glucogenic Leucine Isoleucine Rest of amino acids (14) Lysine Tyrosine Tryptophan Phenylalanine By Henok. G 9  Amino acids & Proteins Although about 300 amino acids exist in cells, only 22 of them are polymerizable into protein structure and are known as protein amino acids, whereas, those that do not occur in proteins are called as non-protein amino acids: Selenocysteine: Selenium can be substituted in place of sulfur of cysteine to form selenocysteine. This amino acid (called as 21st amino acid of proteins) is present at the active site of a number of selenoproteins, e.g., Glutathione peroxidase, Thioredoxin reductase, Deiodinase and Glycine reductase found in archaea, bacteria and eukaryotes. Selenium has long been associated with anti-oxidant activity. A glycoprotein has been isolated from mammalian blood that contains as many as 10 selenium residues. The protein has antioxidant properties and its concentration is found to be decreased in selenium deficiency. Selenium is activated to selenophosphate with the help of an enzyme selenophosphate synthetase. The active selenium then replaces the hydroxyl of serine amino acid to transform it into selenocysteine, Selenocysteine (Sec) is charged on a special tRNA specific for ‗UGA‘ (STOP) codon that is inserted into the growing polypeptide during translation. To be utilized as Sec specifying codon, the UGA should have a stem-loop structure called ‗selenocysteine insertion sequence element‘, present immediately downstream of the codon mostly in the 3‘-UTRs of the mRNA.  Pyrrolysine (See the figure below) is another non-canonical amino acid, which is encoded by another stop codon, ‗UAG‘, in several methanogenic organisms. Pyrrolysine, hence, is termed as the 22nd protein amino acid present at active site of a number of methyl-transferase enzymes in archaea and bacteria. Pyrrolysine is attached to a specific tRNAPylCUA by a specific pyrrolysine-tRNA synthetase (a class II aminoacyl-tRNA synthetase). The enzyme uses ATP to activate pyrrolysine and ligates it to tRNA carrying CUA as the anticodon that complementates with UAG and converts it into a sense codon rather than STOP. The differential usage of that codon depends upon the availability of the pyrrolysine in the medium and/or the substrate of the target enzyme for which pyrrolysine would be incorporated. H2N CH COOH CH2 CH2 CH2 CH3 CH2 HN O Pyrrolysine N Uncommon Amino Acids Also Have Important Functions: In addition to the 20 common amino acids, other less common amino acids also occur, either as constituents of proteins (through modification of common amino acid residues after protein synthesis) or as free metabolites. Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine.  The former is found in plant cell wall proteins, and both are found in collagen, a fibrous protein of connective tissues.  6-N-Methyllysine is a constituent of myosin, a contractile protein of muscle. By Henok. G 10  Amino acids & Proteins  Another important uncommon amino acid is γ-carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other proteins that bind Ca2+ as part of their biological function.  More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein elastin. Selenocysteine is a special case. This rare amino acid residue is introduced during protein synthesis rather than created through a post synthetic modification.  It contains selenium rather than the sulfur of cysteine.  Actually derived from serine, selenocysteine is a constituent of just a few known proteins. Some 300 additional amino acids have been found in cells. They have a variety of functions but are not constituents of proteins.  Ornithine and citrulline deserve special note because they are key intermediates (metabolites) in the biosynthesis of arginine and in the urea cycle. TABLE–1 Properties and Conventions Associated with the Common Amino Acids Found in Proteins By Henok. G 11  Amino acids & Proteins Properties of amino acids I.Physical properties: 1.Solubility: All amino acids are soluble in water, diluted acids and alkalis. 2.Optical activity: All amino acids, except glycine, are optically active, i.e., they contain asymmetric carbon atom (-carbon), thus they can rotate plane-polarized light either to the right or to the left. 3.Absorption of ultraviolet light: Aromatic amino acids (tryptophan, tyrosine and phenylalanine) can absorb ultraviolet light. This is utilized to measure concentrations of amino acids and proteins in solution. II.Chemical properties of amino acids: 1.Reactions due to:-Carboxyl group –COOH; Amino group – NH2; and Radical – R. A.Reactions due to carboxyl group such as:- Reaction with alkalis to form salts Reaction with alcohol to form esters By Henok. G 12  Amino acids & Proteins Formation of primary amines and amides B.Reactions due to amino group such as:-  Reaction with acids to form salts  Reaction with nitrous acid to liberate nitrogen (Van Slyke reaction)  Reaction with CO2 in alkaline medium to form carbamino compound  Reaction with acyl anhydrides or halides such as benzoyl chloride to give acyl derivatives, e.g., reaction with glycine to give hippuric acid that is used to get ride of ammonia in liver cirrhosis, O O O O C Cl C N CH C OH + HCl H H2N CH C OH + H H benzoyl chloride hippuric acid  Reaction with fluorodinitrobenzene to form dinitrophenyl amino acid (DNP- amino acid) used to identify the N-terminal amino acid in any protein and to separate amino acids in a mixture by paper chromatography.  N.B.: Hydrazine can be used to identify the C-terminal amino acid in any protein.  Reaction with ninhydrin to form a blue compound except with proline and hydroxyl proline which contain imino group (–NH) and not amino group give a red color which rapidly changes to yellow color.  It is utilized in chromatographic separation of amino acids. O Ninhydrin Ninhydrin H2N CH C OH R-CHO + CO2 + NH3 Yellow or blue R Aldehyde complex O2 C.Reaction due to the Radical such as:-  Millon's reaction: It is a reaction between phenol group of tyrosine and Millon‘s reagent (mercuric and mercurous nitrates and nitrites) giving red color by heating.  Sakaguchi reaction: It is a reaction between the guanido group of arginine and - naphthol forming a red color.  Xanthoproteic reaction; Pauly reaction; Nitroprusside reaction; & Rosenheim's test used to differentiate amino acids. 2.Amino Acids Can Act as Acids and Bases: Amino acids vary in their acid-base properties and have characteristic titration curves. When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or zwitterion (German for ―hybrid ion‖), shown in Figure AA-5. Each amino acid has a specific pH at which it carries equal negative (-COO-) and positive (NH3+) charges because of equal ionization of its ionizable groups. This means it carries no net charge or isoionic. This pH value is called isoelectric pH or point (pI), and the amino acid form at that pI is termed Zwitterion (zwi = equal).  Definition of isoelectric pH (pI):  It is the pH at which Zwitterion is formed. Its importance is as follows: During separation by electrophoresis of amino acids or proteins pH value should avoided the isoelectric point (I.E.P.). Isoelectric point is also utilized to precipitate an amino acid or a protein from a solution or a mixture. By Henok. G 13  Amino acids & Proteins It is used to identify the compound. FIGURE AA-5 Nonionic and zwitterionic forms of amino acids. The nonionic form does not occur in significant amounts in aqueous solutions. The zwitterion predominates at neutral pH.  A zwitterion can act as either an acid (proton donor):  or a base (proton acceptor):  Substances having this dual nature are amphoteric and are often called ampholytes (from ―amphoteric electrolytes‖).  A simple monoamino monocarboxylic - amino acid (with nonionizable R groups), such as alanine, is a diprotic acid when fully protonated—it has two groups, the - COOH group and the -NH3+ group, that can yield protons: Amino Acids Differ in Their Acid-Base Properties: The shared properties of many amino acids permit some simplifying generalizations about their acid-base behaviors. First, all amino acids with a single -amino group, a single -carboxyl group, and an R group that does not ionize have titration curves resembling that of glycine (Fig. AA-6).  These amino acids have very similar, although not identical, pKa values: pKa of the - COOH group in the range of 1.8 to 2.4, and pKa of the -NH3+ group in the range of 8.8 to 11.0 (Table–1). By Henok. G 14  Amino acids & Proteins FIGURE AA-6 Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25 oC. The ionic species predominating at key points in the titration are shown above the graph. The shaded boxes, centered at about pK1 = 2.34 and pK2 = 9.60, indicate the regions of greatest buffering power.  Second, amino acids with an ionizable R group have more complex titration curves, with three stages corresponding to the three possible ionization steps; thus they have three pKa values.  The additional stage for the titration of the ionizable R group merges to some extent with the other two. The titration curves for two amino acids of this type, glutamate and histidine, are shown in Figure AA-7.  The isoelectric points reflect the nature of the ionizing R groups present. For example, glutamate has a pI of 3.22, considerably lower than that of glycine.  This is due to the presence of two carboxyl groups, which, at the average of their pKa values (3.22), contribute a net charge of -1 that balances the +1 contributed by the amino group.  Similarly, the pI of histidine, with two groups that are positively charged when protonated, is 7.59 (the average of the pKa values of the amino and imidazole groups), much higher than that of glycine.  Finally, as pointed out earlier, under the general condition of free and open exposure to the aqueous environment, only histidine has an R group (pKa = 6.0) providing significant buffering power near the neutral pH usually found in the intracellular and extracellular fluids of most animals and bacteria (Table –1). By Henok. G 15  Amino acids & Proteins FIGURE AA-7 Titration curves for (a) glutamate and (b) histidine. The pKa of the R group is designated here as pKR. Peptides and Proteins We now turn to polymers of amino acids, the peptides and proteins.  Biologically occurring polypeptides range in size from small to very large, consisting of two or three to thousands of linked amino acid residues.  Our focus is on the fundamental chemical properties of these polymers. Peptides Are Chains of Amino Acids: Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide.  Such a linkage is formed by removal of the elements of water (dehydration) from the -carboxyl group of one amino acid and the -amino group of another (Fig. AA-8). By Henok. G 16  Amino acids & Proteins  Peptide bond formation is an example of a condensation reaction, a common class of reactions in living cells. FIGURE AA-8 Formation of a peptide bond by condensation. The - amino group of one amino acid (with R2 group) acts as a nucleophile to displace the hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced. At physiological pH, the reaction shown does not occur to any appreciable extent. Three amino acids can be joined by two peptide bonds to form a tripeptide; similarly, amino acids can be linked to form tetrapeptides, pentapeptides, and so forth.  When a few amino acids are joined in this fashion, the structure is called an oligopeptide. When many amino acids are joined, the product is called a polypeptide. Proteins may have thousands of amino acid residues.  Although the terms ―protein‖ and ―polypeptide‖ are sometimes used interchangeably, molecules referred to as polypeptides generally have molecular weights below 10,000, and those called proteins have higher molecular weights. Figure AA-9 shows the structure of a pentapeptide. As already noted, an amino acid unit in a peptide is often called a residue (the part left over after losing a hydrogen atom from its amino group and the hydroxyl moiety from its carboxyl group).  In a peptide, the amino acid residue at the end with a free -amino group is the amino-terminal (or N-terminal) residue; the residue at the other end, which has a free carboxyl group, is the carboxyl-terminal (C-terminal) residue. Although hydrolysis of a peptide bond is an exergonic reaction, it occurs slowly because of its high activation energy. As a result, the peptide bonds in proteins are quite stable, with an average half-life (t1/2) of about 7 years under most intracellular conditions. By Henok. G 17  Amino acids & Proteins FIGURE AA-9 The pentapeptide serylglycyltyrosylalanylleucine, or Ser–Gly– Tyr–Ala–Leu. Peptides are named beginning with the amino terminal residue, which by convention is placed at the left. The peptide bonds are shaded in yellow; the R groups are in red. Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes: No generalizations can be made about the molecular weights of biologically active peptides and proteins in relation to their functions.  Naturally occurring peptides range in length from two to many thousands of amino acid residues. Even the smallest peptides can have biologically important effects.  Consider the commercially synthesized dipeptide L-aspartyl-L-phenylalanine methyl ester, the artificial sweetener better known as aspartame or NutraSweet. Many small peptides exert their effects at very low concentrations.  For example, a number of vertebrate hormones are small peptides. These include: Oxytocin (nine amino acid residues), which is secreted by the posterior pituitary and stimulates uterine contractions; Bradykinin (nine residues), which inhibits inflammation of tissues; and Thyrotropin-releasing factor (three residues), which is formed in the hypothalamus and stimulates the release of another hormone, thyrotropin, from the anterior pituitary gland.  Glutathione (-glutamyl, cysteinyl, Glycine): It is a tripeptide formed of 3 amino acids: -glutamic, cysteine and glycine. OH O C H2 H2 O O O CH C C C HN CH C HN CH C OH NH2 CH2 H OH O C S H2 H2 O O O 2H CH C C C HN CH C HN CH C OH S NH2 CH2 H NH2 CH2 H H2 H2 SH CH C C C HN CH C HN CH C OH 2 Glutathione, reduced O C O O O OH Glutathione, oxidized Functions of glutathione: By Henok. G 18  Amino acids & Proteins 1.It has a role in absorption of amino acids. 2.It activates many enzymes. 3.It inactivates insulin hormone, by breaking its disulfide bonds. 4.It protects the cell membrane from damage, e.g., prevents hemolysis of erythrocytes. 5.It prevents rancidity of fat (or lipid peroxidation) as it acts as antioxidant.  Some extremely toxic mushroom poisons, such as amanitin, are also small peptides, as are many antibiotics. Slightly larger are small polypeptides and oligopeptides such as: The pancreatic hormone insulin, which contains two polypeptide chains, one having 30 amino acid residues and the other 21. Glucagon, another pancreatic hormone, has 29 residues; it opposes the action of insulin. Corticotropin is a 39-residue hormone of the anterior pituitary gland that stimulates the adrenal cortex. How long are the polypeptide chains in proteins? As Table –2 shows, lengths vary considerably.  Human cytochrome c has 104 amino acid residues linked in a single chain;  Bovine chymotrypsinogen has 245 residues.  At the extreme is titin, a constituent of vertebrate muscle, which has nearly 27,000 amino acid residues and a molecular weight of about 3,000,000.  The vast majority of naturally occurring proteins are much smaller than this, containing fewer than 2,000 amino acid residues. Some proteins consist of a single polypeptide chain, but others, called multisubunit proteins, have two or more polypeptides associated noncovalently (Table –2).  The individual polypeptide chains in a multisubunit protein may be identical or different.  If at least two are identical the protein is said to be oligomeric, and the identical units (consisting of one or more polypeptide chains) are referred to as protomers. Hemoglobin, for example, has four polypeptide subunits: two identical  chains and two identical β chains, all four held together by noncovalent interactions. Each  subunit is paired in an identical way with a β subunit within the structure of this multisubunit protein, so that hemoglobin can be considered either a tetramer of four polypeptide subunits or a dimer of β protomers. A few proteins contain two or more polypeptide chains linked covalently.  For example, the two polypeptide chains of insulin are linked by disulfide bonds.  In such cases, the individual polypeptides are not considered subunits but are commonly referred to simply as chains. We can calculate the approximate number of amino acid residues in a simple protein containing no other chemical constituents by dividing its molecular weight by 110.  Although the average molecular weight of the 20 common amino acids is about 138, the smaller amino acids predominate in most proteins. By Henok. G 19  Amino acids & Proteins  If we take into account the proportions in which the various amino acids occur in proteins (Table –1), the average molecular weight of protein amino acids is nearer to 128.  Because a molecule of water (Mr 18) is removed to create each peptide bond, the average molecular weight of an amino acid residue in a protein is about 128 - 18 = 110. TABLE –2 Molecular Data on Some Proteins: Polypeptides Have Characteristic Amino Acid Compositions: Hydrolysis of peptides or proteins with acid yields a mixture of free -amino acids.  When completely hydrolyzed, each type of protein yields a characteristic proportion or mixture of the different amino acids. The 20 common amino acids almost never occur in equal amounts in a protein.  Some amino acids may occur only once or not at all in a given type of protein; may others occur in large numbers.  Table –3 shows the composition of the amino acid mixtures obtained on complete hydrolysis of bovine cytochrome c and chymotrypsinogen, the inactive precursor of the digestive enzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind of amino acid they contain. TABLE –3 Amino Acid Compositions of Two Proteins: By Henok. G 20  Amino acids & Proteins Some Proteins Contain Chemical Groups Other Than Amino Acids: Many proteins, for example the enzymes ribonuclease A and chymotrypsinogen, contain only amino acid residues and no other chemical constituents; these are considered simple proteins. However, some proteins contain permanently associated chemical components in addition to amino acids; these are called conjugated proteins.  The non–amino acid part of a conjugated protein is usually called its prosthetic group. Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups (Table–4); for example, lipoproteins contain lipids, glycoproteins contain sugar groups, and metalloproteins contain a specific metal.  A number of proteins contain more than one prosthetic group.  Usually the prosthetic group plays an important role in the protein‘s biological function. TABLE –4 Conjugated Proteins: By Henok. G 21  Amino acids & Proteins Bonds participating in the protein structure: 2 classes of strong bonds (peptide and disulfide) and 3 classes of weak bonds (hydrogen, hydrophobic and electrostatic bonds) generally stabilize Protein structure. I.Strong bonds: 1.Peptide bonds (primary bond): A peptide bond is a covalent bond formed by a reaction between amino group of one amino acid and a carboxylic group of the next amino acid with the loss of H 2O that required ATP. It is the strongest bond in the protein molecule that resists denaturation and is called primary because it is the only bond in the primary structure of the polypeptide. 2.Disulfide bonds (secondary bond): The disulfide bond is formed between the SH groups of two cysteine residues within same (intra-chain) or two different polypeptide chains (inter-chain).  It maintains secondary structure of a peptide chain or connects two polypeptide chains together in the tertiary structure.  It follows the peptide bond in strength but liable to denaturation. O O R HN CH C R R HN CH C R peptide chain CH2 CH2 2H SH S two cysteine disulfide bond residues SH S CH2 CH2 R HN CH C R peptide chain R HN CH C R O O II.Weak bonds (secondary bonds): 1.Hydrogen bonds: Hydrogen bond is a weak bond formed between the hydrogen atom of –NH of a peptide bond on one peptide chain and the oxygen of C=O of another peptide bond on an adjacent peptide chain or a loop belongs to same peptide chain. R CH C N peptide chain O H hydrogen bond H O N C peptide chain CH R By Henok. G 22  Amino acids & Proteins 2.Hydrophobic bonds: The non polar side chains of neutral amino acids tend to associate in hidden core of protein molecule away from solvent. O R O R HN CH C HN CH C HN CH C HN CH C R O R O 3.Electrostatic bonds: These are salt bonds formed between oppositely charged groups in the side chains of amino acids e.g. -amino group of lysine and the carboxyl group of asparatic acid. O O H2N CH C OH H2N CH C OH CH2 CH2 Asparatic acid CH2 C O Lysine CH2 O- CH2 NH3+ Electrostatic attraction There Are Several Levels of Protein Structure: Four levels or orders of organization of protein structure are commonly defined: primary, secondary, tertiary and quaternary structures (Fig. AA-10).  This complication gives the molecule its functional domain to explain its structure- function requirements that if changes due to mutation will give non-functional protein and, therefore, a disease. 1.Primary structure: It is the linear form of the polypeptide illustrating the total number, chemical nature, and linear order of all of the amino acid residues in the polypeptide chain or chains of a protein and position of disulfide bonds if present.  The peptide bonds (primary bond) are responsible for the primary structure.  It is dedicated by the genetic code in specific gene and it determines the final conformational shape of a polypeptide. Substitution of a single amino acid for another in the linear sequence of amino acids in the polypeptide chain may reduce or abolish biologic activity of the protein with potentially serious consequences.  An example is Sickle Cell Anemia where the normal glutamic acid (polar) at position 6 of the -chains is replaced by a valine (non-polar). 2.Secondary structure: It is the fine folding of polypeptide chain into specific regular coiled structure such as helix or -pleated sheet or irregular random coiling held together by hydrogen, ionic and disulfide bonds. By Henok. G 23  Amino acids & Proteins  It is due to the interaction of amino acids located very close to each other.  This gives the polypeptide chain a specific space conformation. 3.Tertiary structure: It is the final three-dimensional form due to the more complicated course folding and super-folding of the polypeptide chain in its secondary level into globular or fibrous form of different size.  It is due to interaction of amino acids located far apart.  It is the biologically active conformation of the polypeptide and therefore, is the most liable to denaturation.  The bonds stabilizing the tertiary structure are disulfide bonds, hydrogen bonds, ionic bonds, van-der-Waal‘s attraction and hydrophobic interaction (all are secondary bonds).  According to it the protein is classified as fibrous or globular. Secondary and tertiary structure of the protein is determined by the amino acid structure of the primary polypeptide chain. 4.Quaternary structures: Proteins consist of two or more polypeptide chains in their tertiary structure united by forces other than peptide bonds are said to possess a quaternary structure.  Quaternary structure therefore, is the positional relationship between individual polypeptides associating to form one protein molecule.  The bonds responsible for the quaternary structure are as those in the tertiary structure.  It is the most liable to denaturation.  If this structure alternate between two states upon binding to other proteins or a prothestic group, the protein is called an allosteric protein.  Examples are globin of hemoglobin and lactate dehydrogenase each is composed of 4 polypeptide chains.  Apotransferrin is composed of 24 identical subunits and Creatine kinase is composed of two subunits. By Henok. G 24  Amino acids & Proteins Random coils Subunit  -Sheet -helix  -Sheet -helix Random coils Subunit Quaternary protein structure  Finally, different proteins – each of these proteins alone has all 4 orders of structure – aggregate into macro-molecular complexes as exemplified by cell membrane proteins, electron transport proteins and fatty acid synthase enzyme. FIGURE AA-10a Levels of structure in proteins. The primary structure consists of a sequence of amino acids linked together by peptide bonds and includes any disulfide bonds. The resulting polypeptide can be coiled into units of secondary structure, such as an  helix. By Henok. G 25  Amino acids & Proteins The helix is a part of the tertiary structure of the folded polypeptide, which is itself one of the subunits that make up the quaternary structure of the multisubunit protein, in this case hemoglobin. By Henok. G 26  Amino acids & Proteins ▲ Figure AA-10b Overview of protein structure and function These functions and others arise from specific binding interactions and conformational changes in the structure of a properly folded protein. By Henok. G 27  Amino acids & Proteins Classification of Proteins I.According to the biological importance of the protein: 1.Proteins of high biological value: These are all proteins of animal origin (with a few exceptions) and some proteins of plant origin that contain all the 10 essential amino acids in well balanced amounts and are easily digestible.  Examples of animal proteins include; milks and its products, egg, liver, fishes, red and white meats.  Examples of the few plant proteins of high biological value are lentils and broad beans. 2.Proteins of low biological value: These are proteins that are deficient in one or more of the essential amino acids or containing very little amount of one of them or are indigestible.  Most of plant protein are of low biological value and a very few animal proteins are also of low biological value such are collagen because is deficient in tryptophan and cysteine and keratins because they are indigestible. This does not imply that a person should eat only a protein of high biological value to avoid deficiency of essential amino acids, but this can be also avoided by eating two or more proteins of low biological value that complete each other‘s deficiency. II.According to the axial ratio of the protein molecule: Studies on the shape of the protein molecule using ultramicroscope indicates that there are two types of proteins in nature:  Fibrous proteins.  Globular proteins. 1.Fibrous proteins: They have an axial ratio of more than 10.  Axial ratio = Length/Width of the protein molecule.  They are fairly stable proteins. Examples, a.Keratin proteins in hairs, wool, skin, and most cells.  In its native state, it is present in the form of coiled polypeptide chains called - keratin.  It can be stretched by denaturation forming -keratin. b.Myosin is the major protein of muscles.  During muscle relaxation it is called -myosin but during muscle contraction, it undergoes a change in its structure and it becomes -myosin. 2.Globular proteins: Their axial ratio is less than 10.  Their peptide chains are folded or coiled on themselves in a very compact manner.  They are less stable than fibrous proteins. Examples are albumin, globulins, and insulin. III.According to the composition of the Protein: There are 3 main groups: Simple proteins By Henok. G 28  Amino acids & Proteins Conjugated proteins Derived proteins A.Simple Proteins: These are proteins which on hydrolysis produce amino acids only.  Simple proteins are subdivided according to their physical properties, solubility, molecular weight and amino acid composition into: Albumins, Globulins, Protamines, Histones, Gliadins or Prolamines, Glutelins, & Scleroproteins (Albuminoids; such as: Elastins, Collagens, Reticulins, Keratins). Albumins Albumins are much distributed in nature. They are usually associated with globulins. Albumins are characterized by giving positive heat coagulation test. Coagulation happens at about 75oC.  Slight acidity accelerates the coagulation, while alkalinity retards it. The heat coagulation test is one of the most important investigations done for the detection of albumin in urine in kidney problems.  Albumins are readily soluble in distilled water and also soluble in very dilute salt solutions, alkalis and acids.  They are not precipitated by half saturation with neither magnesium sulfate nor sodium chloride, but precipitated by full-saturation with ammonium sulfate. Albumins are proteins of high biological value since it is not deficient in any of the essential amino acids but deficient in glycine.  They give all the color tests of proteins. They are acidic proteins at pH 7.4 and have pI of 4.7.  Examples: Serum albumin present in blood, lactalbumin of milk, ovalbumin of egg, leucosin of cereals and legumelin of legumes such as peas. Pepsin is probably an albumin. Globulins Globulins are always present associated with albumins and are also positive with coagulation test.  Euglobulins or true globulins are soluble in saline and insoluble in distilled water. Pseudoglobulins (false globulin) are soluble in distilled water.  Like albumins, they are soluble in dilute acids and alkalis. Globulins are of high biological value and it is not deficient in any of the essential amino acids. Globulin is precipitated by half saturation with ammonium sulfate or full saturation with NaCl or MgSO4.  This is due to the fact that globulins are bigger in molecular weight than albumins. The most important members of globulins are serum globulins.  By electrophoresis blood globulin was found to be of three, B and  types, which possess different molecular weights.  Myosin of muscle, thyroglobulin of the thyroid gland, antibodies of the blood, phosphatase enzyme, urease, trypsin, ceruloplasmin, transferrin, chymotrypsin and legumin of peas are all examples of globulins. By Henok. G 29  Amino acids & Proteins Albumin Globulin - Soluble in water and salt solution - Soluble in salt solution. - Coagulated by heat. - The same as albumin -They are of high biological value: -The same as albumin. - Contain all essential amino acids. -The same as albumin. - Easily digested. -The same as albumin. - M.W.: 68 KDa. - M.W.: 150 KDa. -Precipitated by full saturation with -Precipitated by half saturation with ammonium sulfate. ammonium sulfate. -They are present in: serum albumin, They are present in: serum globulin, egg albumin, milk: lactalbumin. milk Lactglobulin, egg globulin. - It functions as transporting protein for - It functions in transport also but its elements, vitamins, and hormones other major function is being antibodies. than keeping blood osmosis. Scleroproteins (Albuminoids) Scleroproteins are characterized by their extreme insolubility in water, dilute acids and the most common reagents.  They are strong fibrous structural proteins.  Their main function is the protection of the body that are rich in sulfur-containing amino acids and hence disulfide bonds.  Hairs, nails, natural silk and connective tissues, contain scleroproteins.  Albuminoids are never present in plants. The main important groups of scleroproteins are: Elastins Collagens Reticulins Keratins Elastins: They are present in the yellow fibers of the connective tissues which are mainly present in lungs, uterine wall during pregnancy, tendons and ligaments.  Elastins are also present in the elastic tissues of tendons and big arteries.  It is rich in alanine, leucine, valine and proline but deficient in cysteine, methionine, lysine and histidine. They are insoluble in water, dilute acids, dilute alkalis and saline.  Boiling scleroproteins with strong acids or strong alkalis or their digestion by elastase leads to their hydrolysis to free amino acids. Collagens: They are present in white fibrous connective tissues, tendons and bones. By Henok. G 30  Amino acids & Proteins  Collagen is insoluble in water, dilute acids and alkalis. From this point of view it is similar to elastins.  Collagen is resistant to peptic and trypsin digestion, but when boiled for a long time with water, dilute acids or alkalis, it changes to gelatin.  Thus, gelatin is a derived protein obtained from the partial hydrolysis of collagen.  Both gelatin and collagen are of little nutritive value because they contain about 40% of the non-essential amino acid glycine. Collagen is rich in glycine, proline and hydroxy proline but low in sulfur containing amino acids. Glycine, proline and hydroxy proline form about 2/3 of the total amount of amino acids present in the collagen molecule.  Collagen is deficient in tryptophan and cysteine.  Collagen differs from keratins in containing much less sulfur. Gelatins:  It is the product of prolonged boiling of collagen in water. It is easily digested and has the property of forming a gel on cooling (gel formation).  Gelatin is a very good diet for patients because it is an appetizer and easily digested.  Gelatin is deficient in certain essential amino acids. So it is not an adequate protein diet, as it is deficient in tryptophan and cysteine and contains very small amounts of methionine (protein of low biological value). Reticulins: Reticulins are present in the reticular tissues such as spleen, liver and lymph glands.  In properties, reticulins are very much similar to elastin and collagen.  Reticulins are unaffected by dilute acids or alkalis. Keratins (or cytokeratins): Keratins are highly insoluble compounds.  They are insoluble in all protein solvents, and are not digestible by proteolytic enzymes (e.g., pepsin and trypsin).  Keratins are hydrolyzed by prolonged boiling with alkalis. The sulfur content of keratin is high.  It is present in the form of cystine, which is responsible for the stability and insolubility of keratins. Most keratins yield histidine, lysine and arginine amino acids on hydrolysis, in a molecular ratio of, 1:4:12, respectively. Barium sulfide is one of the important substances that dissolve keratins.  For this reason this material enters in the formation of cosmetics dealing with removal of hair. Keratins are present in hairs, nails and superficial layer of the skin. They form the intermediate filaments of the cytoskeleton in the epithelial cells where their expression is stable to trace the origin of these cells. They composed of more than 20 types of polypeptides.  The cystine content is about 14%. This is why it is a good source of cystine. B.Conjugated proteins:  On hydrolysis, they give amino acids and prosthetic group, i.e., a non-protein group. They include:  Phosphoproteins, lipoproteins, glycoproteins, metalloproteins, chromoproteins, & nucleoprotein. By Henok. G 31  Amino acids & Proteins 1.Phosphoproteins: These are proteins conjugated with phosphate. Phosphate is attached to OH group of serine, tyrosine or threonine present in protein. They are found in: a. Casein: The main milk protein. b. Vitellin: It is present in egg yolk. 2.Lipoproteins: These are proteins conjugated with lipids converting them into water soluble substances.  Examples: Plasma lipoproteins: see lipid chemistry. 3.Glycoproteins: These are proteins conjugated with carbohydrates in varying amounts attached as short or long chains.  Examples: Mucous secretion of gastrointestinal tract and Glycoproteins of cell wall. 4.Metalloproteins: These are proteins conjugated with metals such as; 1.Iron: e.g., ferritin is intracellular iron-binding protein and Transferrin is an iron-binding transport protein in the blood. 2.Zinc: e.g., Insulin hormone present in crystals containing zinc. 3.Copper: e.g., Ceruloplasmin: is a protein present in blood. It is responsible for the oxidation of Fe2+ ions to Fe3+ ions. 5.Chromoproteins: These are proteins conjugated with colored pigment.  Example; Hemoglobin and cytochrome enzyme present in mitochondria contains haem pigment, which is red in color. 6.Nucleoprotein: These are proteins (protamines or histones) conjugated with nucleic acids (DNA or RNA).  Examples; Chromosomes: These are proteins conjugated with DNA.  Ribosomes: They are proteins conjugated with RNA. C.Derived Proteins: They include: A.Denatured protein: e.g., coagulated albumin or globulin. B.Hydrolytic product of protein: e.g., Protein  Proteoses  Peptone  Polypeptide. 1.Proteoses, they are soluble in water, not coagulable and precipitated by saturated salt solution. 2.Peptones: are soluble in water and not coagulable by heat. 3.Peptides: are soluble in water and salt solution, not coagulable by heat. They are formed from 2 or more amino acids. By Henok. G 32