1. CHAPER 24 AMINO ACIDS AND PROTEINS 24.1 INTRODUCTION Of the three groups of biopolymers, protein have the most diverse function. Most of its molecular weights are much larger. Their shaps cover a range from the globular protein to the helical coils of a α–keratin. But all proteins have common features. Proteins are polyamides and their monomeric units are about 20 different α-amino acids.

2. Primary structure: the exact sequence of the different α-amino acids along the protein chain. Second and tertiary structure: the folding of the polyamide chain which give rise to higher levels of complexity. Although hydrolysis of natural occurring proteins may yield as many as 22 different amino acids, the amino acids have an important structural feature in common.

3. 24.2 AMINO ACIDS 24.2A STRUCTURES AND NAMES

6. The conversion of cysteine to cystine requires addition comment. it can be reversed by mild reducing agents.

7. 24.2B ESSENTIAL AMINO ACIDS For adult humans there are eight essential amino acids. These are designated with the superscript e in above table. 24.2C AMINO ACIDS AS DIPOLAR IONS Since amino acids contain both a basic group (-NH 2 ) and an acidic group (-COOH), they are amphoteric.

8. In strongly basic solutions all amino present as anions, in acidic solutions they are present as cations. At some intermediate Ph, called isoelectric point(pI) the concentration of the dipolar ion is at its maximum and the concentrations of the anions and cations are equal. As the acidity reaches pH 2.3, one half of the cationic form will be converted to the dipolar ion. As the pH increase to2.3- 9.7 the predominant form will be the dipolar ion. When pH rise to 9.7,the dipolar ion will be half-converted to the anionic form. As pH approached to 14,the anionic form becomes predominant form.

9. If the side chain of an amino acid contains an extra acidic or basic group, then the equilibria are more complex. The isoelectric point (pI) of an amino such as the alanine is the average of pK a 1 and pK a 2. 24.3 LABORATORY SYNTHESIS OF α -AMINO ACIDS A variety of methods have been developed for the laboratory synthesis of α-amino acids. We shall describe here three general methods.

10. 24.3A DIRECT AMMONOLYSIS OF AN α -HALO ACID This method is probably used least often because yields tend to be poor. 24.3B FROM POTASSIUM PHTHALIMIDE This method is a modification of the Gabriel synthesis of amines. the yields are usually high and the products are easily purified.

11. 24.3C THE STRECKER SYNTHESIS Treating an aldehyde with ammonia and hydrogen cyanide produces an α-amino nitrile. Hydrolysis of the nitrile group of the α-amino nitrile converts the latter to an α-amino acid.

12. Mechanism of the first step: 24.3D RESOLUTION OF DL -AMINO ACIDS

13. One interesting method for resolving amino acids is based on the use of enzymes called deacylases. 24.3E STEREOSELECTIVE SYNTHESIS OF AMINO ACIDS

14. Producing only the naturally occurring L -amino acid has been realized through the use of chiral hydrogenation catalysts from transition metals. One of which is called “(R)-prophos”. Hydrolysis of N-acetyl group under this chiral rhodium complex yields L -alanine. Because the hydrogenation catalyst is chiral, it transfers its hydrogen atoms in a stereoselective way. This type of reaction is called asymmetric synthesis.

15. 24.4 ANALYSIS OF AMINO ACID MIXTURES Enzymes can cause α-amino acids to polymerize through the elimination of water:

16. The –CO-NH- linkage between the amino acids is called a peptide bond. Amino acid when joined in this way, are called amino acid residues. The polymers that contains 2,3, a few, or many amino acid residues are called dipeptides, tripeptides, oligopeptides, and polypeptides, respectively. Polypeptides are linear polymers. The free group and the free group are called the N-terminal and the C-terminal Residues respectively.

17. The automatic amino acid analyzers are based on the use of insoluble polymers containing sulfonate groups, called cation-exchange resins.

18. If the mixture of amino acids pass through a column which is washed with a buffered solution at a given pH, The individual amino acids will move down the column at different rates and ultimately separated. 24.5 AMINO ACID SEQUENCE OF POLYPEPTIDES AND PROTEINS The different amino acid sequences of a protein which compose of 20 different amino acids in a single chain of 100 residues are Amazing large. They are The methods of determining the amino acid sequence include Terminal residue analysis, partial hydrolysis and so on.

19. 24.5A TERMINAL RESIDUE ANALYSIS One very useful method for determining the N-terminal amino acid residue, called the Sanger method, is based on the use of 2,4- dinitrofluorobenzene (DNFB).

20. A second method of N-terminal analysis is the Edman degradation. This method offers an advantage over the Sanger method in that it Moves the N-terminal residue and leaves the remainder of the peptide Chain intact.

22. C-terminal residues can identified through the use of digestive enzymes called carboxypeptidases. These enzymes specifically catalyze the hydrolysis of the amide bond of the amino acid residue containing a free –COOH group, liberating it as a free amino acid. 24.5B PARTIAL HYDROLYSIS Break the polypeptide chain into small fragments, then examine the structure of these smaller fragments to determine the original polypeptide. For example: We are given a pentapeptide known to contain valine(two residues), lucine, Histidine, and phenylalanine. Then the molecular formular: Val 2, Leu, His, Phe

23. By using DNFB and carboxypeptidase we discover that valine and leucine are the N-terminal and C-terminal, respectively. Val ( Val, His, Phe) Leu We then subject the pentapeptide to partial acid hydrolysis and obtain the following dipeptides. Val·His + His·Val + Val·Phe + Phe·Leu The points of overlap of the dipeptides tell us that the original Pentapeptide must have been the following: Val· His· Val· Phe· Leu

24. 24.6 PRIMARY ATRUCTURES OF POLYPEPTIDES AND PROTEINS The covalent structure of a protein or polypeptide is called primary structure. Chemists have had remarkable success in determining the primary structure. 24.6A OXYTOCIN AND VASOPRESSIN Oxytocin and vasopressin are two rather polypeptides with strikingly similar structures. But these two polypeptides have quite different physiological effects. Oxytocin occurs only in the female of a species and stimulates uterine contraction during childbirth. Vasopressin occurs in male and female. Its major function is as an antidiuretic.

25. 24.6B INSULIN Insulin, a hormone secreted by the pancreas, regulates glucose metabolism. Bocine insulin has a total of 51 amino acid residues in two poly- peptide chains, called A and B chains. These chains are joined by two disulfide linkage. Human insulin differs from bovine insulin at only three amino acids residues. Insulin from most mammals has a similar structure. 24.6C OTHER POLYPEPTIDES AND PROTEINS

26. Successful sequential analyses have now been achieved with hundreds of other polypeptides and proteins including the following: (1)Bovine ribonuclease. (2) human hemoglobin. (3) bovine trypsinogen and chymotrypsinogen. (4) gamma globulin. 24.7 POLYPEPTIDE AND PROTEIN SYNTHESIS We must first “activate” the carboxyl group of an acid by converting it to an anhydride or acid chloride and then allow it react with an amine. But when both the acid group and the amino group are present in the same molecular, the problem becomes more complicate.

27. 24.7A PROTECTING GROUPS We must “protect” the amino group by converting it to some other group of low nucleophilicity-one that will not react with a reactive acyl derivative. Then remove the protecting group. The reagents are benzyl chloroformate and di-tert-butyl carbonate: Both reagents react with the following amino group to form derivatives that are unreactive toward further acylation.

29. Remove of the benzyl group with hydrogen and a catalyst depends on the fact that benzyl-oxygen bonds are weaker and are subject to hydrogenolysis at low temperatures. 24.7B ACTIVATION OF THE CARBOXYL GROUP A much better method is to convert the carboxyl group of the “protected” amino acid to a mixed anhydride using ethyl chloro- Formate.

30. The mixed anhydride can be used to acylate another amino acid and form a peptide linkage. Dicyclohexylcarbodiimide can also be used to activate the carboxyl group of an amino acid. 24.7C PEPTIDE SYNTHESIS The principle involve here can,of course, be extended to the synthesis of much longer polypeptide chains.

32. The Merrifield method for automated synthesis: 24.7D AUTOMATED PEPTIDE SYNTHESIS

33. 24.8 SECONDARY AND TERTIARY STUCTURES OF PROTEINS 24.8A SECONDARY STRUCTURE

34. The secondary structure of a protein is defined by the local Confor- mation of its polypeptide backbone. These local conformation have come to be specified terms of regular folding patterns. Polypeptide chain of a natural protein can interact with itself two major ways: through formation of a β-pleated sheet and an α helix.

35. Fully extended polypeptide chains could conceivably form a flat- sheet structure (above). Slight rotation of bonds can transform a flat-sheet structure into the β-pleated sheet or β configuration. The α helix structure is a right-handed helix with 3.6amino acid residues per turn in naturally occurring. It is the predominant structure of the polypeptide.α Helices and pleated sheets account for only about one half of the average globular protein. The remaining polypeptide segments have what is called a coil or loop conformation.

36. 24.8B TERTIARY STRUCTURE The tertiary structure of a protein is its three-dimensional shape that arises from further foldings of its polypeptide chains, foldings superimposed on the coils of the α helixes. 24.9 INTRODUCTION TO ENZYMES Enzymes have the ability to bring about vast increases in the rates of reaction. Enzymes also show remarkable specificity for their reactants and for their products. The enzyme and the substrate combine to form an enzyme-substrate complex.

37. Almost all enzymes are proteins. Reactions catalyzed by enzymes are completely stereospecific, and this specificity comes from the way enzymes bind their substrates. Some enzymes will accept only one compound as its substrate, others will accept a range of compounds with similar groups. Inhibitor: a compound that can alter the activity of an enzyme. competitive inhibitor: a compound that competes directly with the substrate for the active site. Some enzymes require the presence of a cofactor. Others may require the presence of an organic molecule called a coenzyme. Many of the water-soluble vitamins are the precursors of coenzymes.

38. 24.10 LYSOZYME: MODE OF ACTION OF AN ENZYME Lysozyme is made up of 129 amino acid residues. Three short segments of the chain between residues 5-15, 24-34, 88-96 have the structure of an α helix; the residues between 41-45, and 50-54 form pleated sheets; and a hairpin turn occurs at residues 46-49. The remaining polypeptide segments of lysozyme have a coil or loop conformation.

39. Lysozyme’s substrate is a polysaccharide of amino sugar that makes up part of the bacterial cell wall. 24.11 SERINE PROTEASES Serine proteases: the digestive enzymes secreted by the pancreas into the small intestines to catalyze the hydrolysis of peptide bonds. The digestive enzymes includes chymotrypsin, trypsin, and elastin. The catalytic triad of chymotrypsin cause cleavage of a peptide bond by acylation of the serine residue 195 of chymotrysin. Near the active site is a hydrophobic binding site that accommodates nonpolar side chains of the protein.

42. Regeneration of the active site of chymotrypsin. Water causes hydrolysis of the acyl-serine bond. Compounds such as diisopropylphosphofluoridate (DIPF) that irreversibly inhibit serine proteases. It has been shown that they do this by reacting only with Ser 195.

43. 24.12 HEMOGLOBIN: A CONJUGATED PROTEIN Hemoglobin: a protein can carry oxygen.

44. The iron of the heme group is in the 2+ oxidation state and it forms a coordinate bond to a nitrogen of the imidazole group of histidine of the polypeptide chain. This leaves one valence of the ferrous ion combine with oxygen as follows: When the heme combing with oxygen the ferrous ion does not become readily oxidized to the ferric state.