Amino Acids and Proteins

Chapter 36

Amino Acids and Proteins

Introduction

• The name protein is taken from the Greek proteins, which means first. This name is well chosen. Of all chemical compounds, proteins must a-lost certainly be ranked Jirsa, for they are the substance of life.

• Proteins make up a large part of the animal body, they hold it together, and they Lun it. They are four.vl in all living cells. They are the principal material of skin, muscle, tendons, nerves, and blood, of enzymes, antibodies, and many hormones.

• Chemically, proteins are haji polymers. They are polyamides, and the monomers from which they are derived are the a-amino carboxylic acids. A single protein molecule contains hundreds or even thousands of amino acid units; these units can be of twenty-odd different kinds. The number of different combinations, that is, the number of different protein molecules that are possible, is almost infinite. It is likely that tens of thousands of different proteins are required to make up and run an animal body; and this set of proteins is not identical with the set required by an animal of a different kind.

• In this chapter we shall look first at the chemistry of the amino acids, and then briefly at the proteins that they make up. Our chief purpose will be to see the ways in which the structures of these enormously complicated molecules are being worked out, and how, in the last analysis, all this work rests on the basic principles of organic structural theory: on the concepts of bond angle and bond length, group size and shape, hydrogen bonding, resonance, acidity and basicity, optical activity, configuration and conformation.

Structure of amino acids

• Table 36.1 gives the structures and names of 26 amino acids that have been found in proteins. Certain of these (marked e) are the essential amino acids, which must be fed to young animals if proper growth is to take place; these particular amino acids evidently cannot be synthesized by the animal from the other materials in its.

• We see that all are alpha-amino carboxylic acids; in two cases (proline and hydroxyproline) the amino group forms part of a pyrrolidine ring. This common feature gives the amino acids a common set of chemical properties, one of which is the ability to form the long polyamide chains that make up proteins. It is on these common chemical properties that we shall concentrate.

• In other respects, the structures of these compounds vary rather widely. In addition to the carboxyl group and the amino group alpha to it. some amino acids contain a second carboxyl group (e.g., aspartic acid or glutamic acid), or a potential carboxyl group in the form of a carboxamide (e.g., asparagine); these are called acidic amino acids. Some contain a second basic group, which may be an amino group (e.g., lysine), a guanidino group (arginine), or the imidazole ring (histidine); these are called basic amino acids. Some of the amino acids contain benzene or heterocyclic ring systems, phenolic or alcoholic hydroxyl groups, halogen or sulfur atoms. Each of these ring systems or functional groups undergoes its own typical set of reactions. 

Amino acids as dipolar ions

• Although the amino acids are commonly shown as containing an amino group and a carboxyl group, H2NCHRCOOH, certain properties, both physical and chemical, are not consistent with this structure:

• (a) In contrast to amines and carboxylic acids, the amino acids are nonvolatile crystalline solids which melt with decomposition at fairly high temperatures.

• (b) They are insoluble in non-polar solvents like petroleum ether, benzene, or ether, and are appreciably soluble in water^ ,/ (c) Their aqueous solutions behave like solutions of substances of high dipole moment. ~ - (d) Acidity and basicity constants are ridiculously low for COOH and NH2 groups. Glycine, for example, has Ka 1/6 x 10~ 10 and KKB = 2.5 x 10~ 12 , whereas most carboxylic acids have AVs of about 10~ 5 and most aliphatic amines have K^s of about 10 ~ 4.

Isoelectric point of amino acids

• What happens when a solution of an amino acid is placed in an electric field depends upon the acidity or basicity of the solution. In quite alkaline solution, H2NCHRCOCr ~< * +H3NCHRCOO- ~< > "H3NCHRCOOH OH- OHII I III

• anions II exceed cations III, and there is a net migration of amino acid toward the anode. In quite acidic solution, cations III are in excess, and there is a net migration of amino acid toward thoughted. If II and III are exactly balanced, there is no net migration; under such conditions any one molecule exists as a positive ion and as a negative ion for exactly the same amount of time, and any small movement in the direction of one electrode is subsequently canceled by an equal movement back toward the other electrode. The hydrogen ion concentration of the solution in which a particular amino acid does not migrate under the influence of an electric field is called the isoelectric point of that amino acid.

• A monoanion monocarboxylic acid, + H3NCHRCOO~, is somewhat more acidic than basic (for example, glycine: Ka = 1.6 x 10- 10 and#& = 2.5 x 10~ 12 ). If crystals of such an amino acid are added to water, the resulting solution contains more of the anion II, H2 NCHRCOO-, than of the cation III, + H3 NCHRCOOH. This "excess" ionization of ammonium ion to amine (l*II 4- H" 1 ") must be repressed, by addition of acid, to reach the isoelectric point, which therefore lies somewhat on the acid side of neutrality (pH 7). For glycine, for example, the isoelectric point is at pH 6.1. 

Configuration of natural amino acid

• From the structures in Table 36.1, we can see that every amino acid except glycine contains at least one chiral center. As obtained by acidic or enzymatic hydrolysis of proteins, every amino acid except glycine has been found optically active. Stereochemical studies of these naturally occurring amino acids have shown that all have the same configuration about the carbon atom carrying the alpha ammo group, and that this configuration is the same as that in L-(~)-glyceraldehyde.

Preparation of amino acid

• Of the many methods that have been developed for synthesizing amino acids, we shall take up only one: adiantum of a-halo acids. Considered in its various modifications, this method is probably the most generally useful, although, like any of the methods, it cannot be applied to the synthesis of all the amino acids. Sometimes an a-chloral or a-bromo acid is subjected to direct ammonolysis with a large excess (Why?) of concentrated aqueous ammonia. For example:

Reactions of amino acids

• The reactions of amino acids are in general the ones we would expect of compounds containing amino and carboxyl groups. In addition, any other groups that may be present undergo their own characteristic reactions.

Peptides. Geometry of the peptide linkage

• Peptides are amides formed by interaction between amino groups and carboxyl groups of amino acids. The amide group, ~ NHCO -, in such compounds is often referred to as the peptide linkage.

• Depending upon the number of amino acid residues per molecule, they are known as dipeptides, tripeptides, and so on, and finally polypeptides. (By convention, peptides of molecular weight up to 10,000 are known as polypeptides and above that as proteins.) For example:

• X-ray studies of amino acids and dipeptides indicate that the entire amide group is flat: carbonyl carbon, nitrogen, and the four atoms attached to them all lie in a plane. The short carbon-nitrogen distance (1.32 A as compared with 1.47 A for the usual carbon-nitrogen single bond) indicates that the carbon-nitrogen bond has considerable double-bond character (about 50%)'; as a result, the angles of the bonds to nitrogen are similar to the angles about the trigonal carbon atom.

• Peptides have been studied chiefly as a step toward the understanding of the much more complicated substances, the proteins. However, peptides are extremely important compounds in their own right: the tripeptide glutathione, for example, is found in most living cells; the nonapeptide oxytocin is a posterior pituitary hormone concerned with contraction of the uterus; a-corticotropin, made up of 39 amino acid residues, is one component of the adrenocorticotropic hormone ACTH. 

Determination of structure of peptides. Terminal residue analysis. Partial hydrolysis

• To assign a structure to a particular peptide, one must know (a) what amino acid residues make up the molecule and how many of each there are, and (b) the sequence in which they follow one another along the.

• To determine the composition of a peptide, one hydrolyzes the peptide (in acidic solution, since alkali causes racemization) and determines the amount of each amino acid thus formed. One of the best ways of analyzing a mixture of amino acids is to separate the mixture into its components by chromatography sometimes, after conversion into the methyl esters (Why?), by gas chromatography.

• From the weight of each amino acid obtained, one can calculate the number of moles of each amino acid, and in this way know the relative numbers of the various amino acid residues in the peptide. At this stage one knows what might be call the "empirical formula" of the peptide: the relative abundance of each amino acid residue in the peptide.

•  Inmates' various modifications, however, the most widely used method of N-terminal residue analysis is one introduced in 1950 by Pehr Edman (of the University of Lund, Sweden). This is based upon the reaction between an amino group and phenyl isothiocyanate to form a substituted thiourea (compare Sec. 32.7). Mild hydrolysis with hydrochloric acid selectively removes the N-terminal residue as the phenylthiohydantpin, which is then identified. The great advantage of

Synthesis of peptides

• Methods have been developed by which a single amino acid (or sometimes a di- or tripeptide) can be polymerized to yield polypeptides of high molecular weight. These products have been extremely useful as model compounds: to show, for example, what kind of x-ray pattern or infrared spectrum is given by a peptide of known, comparatively simple structure.

• Most work on peptide synthesis, however, has had as its aim the preparation of compounds identical with naturally occurring ones. For this purpose, a method must permit the joining together of optically active amino acids to form chains of predetermined length and with a predetermined sequence of residues. Syntheses of this sort not only have confirmed some of the particular structures assigned to natural peptides, but also and this is more fundamental have proved that peptides and proteins are indeed polyamides.

• It was Emit Fischer who first prepared peptides (ultimately one containing 18 amino acid residues) and thus offered support for his proposal that proteins contain the amide link. It is evidence of his extraordinary genius that Fischer played the same role in laying the foundations of peptide and protein chemistry as he did in carbohydrate chemistry.

• The basic problem of peptide synthesis is one of protecting the amino group. In bringing about interaction between the carboxyl group of one amino acid and the amino group of a different amino acid, one must prevent interaction between the carboxyl group and the amino group of the same amino acid. In preparing glycylalanine, for example, one must prevent the simultaneous formation of glycylglycine. Reaction can be forced to take place in the desired way by attaching to one amino acid a group that renders the NH2 unreactive. There are many such protecting groups; the problem is to find one that can be removed later without destruction of any peptide linkages that may have been built up.

Proteins. Classification and function. Denaturation

• Proteins are divided into two broad classes: fibrous proteins, which are insoluble in. water, and globular proteins, which are soluble in water or aqueous solutions of acids, bases, or salts. (Because of the large size of protein molecules, these solutions are colloidal.) The difference in solubility between the two classes is related to a difference in molecular shape, which is indicated in a rough way by their names.

• Molecules of fibrous proteins are long and thread-like and tend to lie side by side to form fibers; in some cases, they are held together at many points by hydrogen bonds. As a result, the intermolecular forces that must be overcome by a solvent are very.

• Molecules of globular proteins are folded into compact units that often approach spheroidal shapes. The folding takes place in such a way that the hydrophobic parts are turned inward, toward each other, and away from water; hydrophilic parts charged groups, for example tend to stud the surface where they are near water. Hydrogen bonding is chiefly intramolecular. Areas of contact between molecules are small, and intermolecular forces are comparatively weak. 

• Only one other class of compounds, the nucleic acids (Sec. 37.7), shows the phenomenon of denaturation. Although closely related to the proteins, polypeptides do not undergo denaturation, presumably because their molecules are smaller and less complex.

Structure of proteins

• We can look at the structure of proteins on a number of levels. At the lowest level, there is the primary structure: the way in which the atoms of protein molecules are joined to one another by covalent bonds to form chains. Next, there is the secondary structure: the way in which these chains are arranged in space to form coils, sheets, or compact spheroids, with hydrogen bonds holding together different chains or different parts of the same chain. Even higher levels of structure are gradually becoming understood: the weaving together of coiled chains to form ropes, for example, or the clumping together of individual molecules to form larger aggregates. Let us look first at the primary structure of proteins.

Peptide chain

• Proteins are made up of peptide chains, that is, of amino acid residues joined by amide linkages. They differ from polypeptides in having higher molecular weights (by convention over 10,000) and more complex structures. The peptide structure of proteins is indicated by many lines of evidence: hydrolysis of proteins by acids, bases, or enzymes yields peptides and finally amino acids; there are bands in their infrared spectra characteristic of the amide group; secondary structures based on the peptide linkage can be devised that exactly fit x-ray.

Side chains. Isoelectric point. Electrophoresis

• To every third atom of the peptide chain is attached a side chain. Its structure depends upon the particular amino acid residue involved: H for glycine, CH3 for alanine, CH(CH3) 2 for valine, CH2C6 H5 for phenylalanine, etc.

• H H H ~N CH C -N-CH-C N -CH-C R 6 R' 6 R" O

• Some of these side chains contain basic groups: NH2 in lysine, or the imidazole ring in histidine. Some side chains contain acidic groups: COOH in aspartic acid or glutamic acid. Because of these acidic and basic side chains, there are positively and negatively charged groups along the peptide chain. The behavior H O H O I I !. N-CH-C -N-CH- C coo- (CH2 )4 + NH3 of a protein in an electric field is determined by the relative numbers of these positive and negative charges, which in turn are affected by the acidity of the solution. At the isoelectric point, the positive and negative charges are exactly balanced, and the protein shows no net migration; as with amino acids, solubility is usually at a minimum here. On the acid side of the isoelectric point, positive charges negative charges and the protein moves to the cathode: on the basic side of the isoelectric point, negative charges exceed positive charges, and the protein moves to the aldol.

Conjugated proteins. Prosthetic groups. Coenzymes

• Some protein molecules contain a non-peptide portion called a prosthetic group; such proteins are called conjugated proteins. The prosthetic group is intimately concerned with the specific biological action of the protein. The prosthetic group of hemoglobin, for example, is hemin. As we see, hemin

• contains iron bound to the pyrrole system known as purpling (compare with the structure of chlorophyll p. 1004). It is the formation of a reversible oxygen-hemin complex that enables hemoglobin to carry oxygen from the lungs to the tissues. Carbon monoxide forms a similar, but more stable, complex; it thus ties up hemoglobin, prevents oxygen transport, and causes death. Hemin is separated from the peptide portion (globin) of the protein by mild hydrolysis; the two units are presumably held together by an amide linkage between a carboxyl group of hemins and an amino group of the polypeptide.

• Many enzymes require cofactors if they are to exert their catalytic effects: metal ions, for example. Organic cofactors are called coenzymes and, if they are covalently bonded to the enzyme, these too are prosthetic groups. The coenzyme biodynamical adenine dinucleotide (NAD), for example, is associated with a number of dehydrogenation enzymes. This coenzyme we see is

• made up of two molecules of D-ribose linked as phosphate esters, the fused heterocyclic system known as adenine, and nicotinamide in the form of a quaternary ammonium salt. In some systems one encounters nicotinamide adenine dinucleotide phosphate (NADP), in which the -OH on C-2 of the left-hand ribose unit of NAD has been phosphorylated. The characteristic biological function of these dehydrogenation enzymes (see, for example, Sec. 37.5) involves conversion of the nicotinamide portion of NAD or NADP into the dihydro structure.

Secondary structure of proteins

• It seems clear that proteins are made up of polypeptide chains. How are these chains arranged in space and in relationship to each other? Are they stretched out side by side, looped and coiled about one another, or folded into independent spheroids?

• Much of our understanding of the secondary structure of proteins is the result of x-ray analysis. For many proteins the x-ray diffraction pattern indicates a regular repetition of certain structural units. For example, there are repeat distances of 7.0 A in silk fibroin, and of 1.5 A and 5.1 A in -keratin of unstretched wool.

• The problem is to devise structures that account for the characteristic x-ray diffraction patterns and are at the same time consistent with what is known about the primary structure: bond lengths and bond angles, planariid of the amide group, similarity of configuration about chiral centers (all L-family), size and sequence of side chains. Of key importance in this problem has been recognition of the stabilizing effect of hydrogen bonds (5-10 kcal per mole per hydrogen bond), and the principle that the most stable structure is one that permits formation of the maximum number of hydrogen bonds. On the basis of the study of simpler compounds, it has been further assumed that the N H---O bond is very nearly linear, hydrogen lying on, or within 20 of, the line between nitrogen and oxygen. In all this work the simultaneous study of simpler, synthetic polypeptides containing only a single kind of amino acid residue has been of great help.

• The progress made on a problem of this size and difficulty has necessarily been the work of many people. Among them is Linus Pauling, of the California Institute of Technology, who received the Nobel Prize in 1954. In 1951 Pauling wrote: "Fourteen years ago Professor Robert B. Corey and I, after we had made a vigorous but unsuccessful attack on the problem of formulating satisfactory configurations of polypeptide chains in proteins, decided to attempt to solve the problem by an indirect method the method of investigating with great thoroughness crystals of amino acids, simple peptides, and related substances, in order to obtain completely reliable and detailed information about the structural characteristics of substances of this sort, and ultimately to permit the confident prediction of precisely described configurations of polypeptide chains in proteins." (Record Chem. Prog., 72, 156-7 (1951).). This work on simple substances, carried on for more than 14 years, gave information about the geometry of the amide group that eventually led Pauling and his co-workers to propose what may well be the most important secondary structure in protein chemistry: the a-helix.

• These chains He side by side to form a flat sheet. Each chain is held by hydrogen bonds to the two neighboring chains (Fig. 36.2). This structure has a repeat distance of 7.2 A, the distance between alternate amino acid residues. (Notice that alternate side chains lie on the same side of the sheet.) However, crowding between side chains makes this idealized flat structure impossible, except perhaps for synthetic Poly glycine.

• Room can be made for small or medium-sized side chains by a slight contraction of the peptide chains:

• When the side chains are quite large, they are best accommodated by a quite different kind of structure. Each chain is coiled to form a helix (like a

• staircase). Hydrogen bonding occurs between different parts of the same chain and holds the helix together. For a-keratin (unstretched wool, hair, horn, nails) Pauling has proposed a helix in which there are 3.6 amino acid residues per turn (Fig. 36.4). Models show that this 3.6-helix provides room for the side chains and allows all possible hydrogen bonds to form. It accounts for the repeat distance of 1.5 A, which is the distance between amino acid residues measured along the axis of the helix. To fit into this helix, all the amino acid residues must be of the same configuration, as, of course, they are; furthermore, their L-configuration requires the helix to be right-handed, as shown. It is becoming increasingly clear that the alpha helix, as it is called, is of fundamental importance in the chemistry of proteins.

•  In 1962, M. F. Perutz and J. C Kendrew of Cambridge University were awarded the Nobel Prize in chemistry for the elucidation of the structure of hemoglobin and the closely related oxygen-storing molecule, myoglobin. Using x-ray analysis and knowing the amino acid sequence (p. 1146), they determined the shape in three dimensions of these enormously complicated molecules: precisely for myoglobin, and very nearly so for hemoglobin. They can say, for example, that the molecule is coiled in an alpha helix for sixteen residues from the Terminal unit, and then turns through a right angle. They can even say vr//r: at the corner there is an aspartic acid residue; its carboxyl group interferes with the hydrogen bonding required to continue the helix, and the chain changes its course. The four folded chains of hemoglobin fit together to make a spheroidal molecule, 64 A x 55 A x 50 A. Four flat heme groups, each of which contains an iron atom that can bind an oxygen molecule, fit into separate pockets in this sphere. When oxygen is being carried, the chains move to make the pockets slightly smaller; Perutz has described hemoglobin as "a breathing molecule." These pockets are lined with the hydrocarbon portions of the amino acids; such a non-polar environment prevents electron transfer between oxygen and ferrous iron and permits the complexing necessary for oxygen transport.