Chapter 26 Biomolecules Amino Acids Peptides and Proteins

Chapter 26: Biomolecules: Amino Acids, Peptides, and Proteins Based on Mc. Murry’s Organic Chemistry, 6 th edition © 2003 Ronald Kluger Department of Chemistry University of Toronto Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 1

Proteins – Amides from Amino Acids n Amino acids contain a basic amino group and an acidic carboxyl group n Joined as amides between the NH 2 of one amino acid and the CO 2 H the next n Chains with fewer than 50 units are called peptides n Protein: large chains that have structural or catalytic functions in biology Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 2

26. 1 Structures of Amino Acids n In neutral solution, the COOH is ionized and the NH 2 is protonated n The resulting structures have “+” and “-” charges (a dipolar ion, or zwitterion) n They are like ionic salts in solution Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 3

The Common Amino Acids n 20 amino acids form amides in proteins n All are -amino acids - the amino and carboxyl are connected to the same C n They differ by the other substituent attached to the carbon, called the side chain, with H as the fourth substituent except for proline n Proline, is a five-membered secondary amine, with N and the C part of a five-membered ring Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 4

Abbreviations and Codes Alanine A, Ala Arginine R, Arg Asparagine N, Asn Aspartic acid D, Asp Cysteine C, Cys Glutamine Q, Gln Glutamic Acid E, Glu Glycine G, Gly Histidine H, His Isoleucine I, Ile Leucine L, Leu Lysine K, Lys Methionine M, Met Phenylalanine F, Phe Proline P, Pro Serine S, Ser Threonine T, Thr Tryptophan W, Trp Tyrosine Y, Tyr Valine V, Val Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 5

Learning the Names and Codes n The names are not systematic so you learn them by using them (They become your friends) n One letter codes – learn them too n If only one amino acid begins with that letter, use it (Cys, His, Ile, Met, Ser, Val) n If more than one begins with that letter, the more common one uses the letter (Ala, Gly, Leu, Pro, Thr) n For the others, some are phonetic: Fenylalanine, a. Rginine, t. Yrosine n Tryp has a double ring, hence W n Amides have letters from the middle of the alphabet (Q – Think of “Qtamine” for glutamine; asparagine -contains N n “Acid” ends in D and E follows (smallest is first: aspartic aci. D, Glutamic acid E) Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 6

Neutral Hydrocarbon Side Chains Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th

-OH, SH (Nucleophiles) and -S-CH 3 Cysteine C, Cys Methionine M, Met Serine S,

Acids and Amides Aspartic acid D, Asp Glutamic Acid E, Glu Asparagine N, Asn

Amines Arginine R, Arg Histidine H, His Lysine K, Lys Tryptophan W, Trp Based

Chirality of Amino Acids n Glycine, 2 -amino-acetic acid, is achiral n In all the others, the carbons of the amino acids are centers of chirality n The stereochemical reference for amino acids is the Fischer projection of L-serine n Proteins are derived exclusively from L-amino acids Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 11

Types of side chains n Neutral: Fifteen of the twenty have neutral side chains n Asp and Glu have a second COOH and are acidic n Lys, Arg, His have additional basic amino groups side chains (the N in tryptophan is a very weak base) n Cys, Ser, Tyr (OH and SH) are weak acids that are good nucleophiles Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 12

Notes on Histidine n Contains an imidazole ring that is partially protonated in neutral solution n Only the pyridine-like, doubly bonded nitrogen in histidine is basic. The pyrrole-like singly bonded nitrogen is nonbasic because its lone pair of electrons is part of the 6 electron aromatic imidazole ring (see Section 24. 4). Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 13

Essential Amino Acids n All 20 of the amino acids are necessary for protein synthesis n Humans can synthesize only 10 of the 20 n The other 10 must be obtained from food Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 14

26. 2 Isoelectric Points n In acidic solution, the carboxylate and amine are in their conjugate acid forms, an overall cation n In basic solution, the groups are in their base forms, an overall anion n In neutral solution cation and anion forms are present n This p. H where the overall charge is 0 is the isoelectric point, p. I Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 15

p. I Depends on Side Chain n The 15 amino acids thiol, hydroxyl groups or pure hydrocarbon side chains have p. I = 5. 0 to 6. 5 (average of the p. Ka’s) n D and E have acidic side chains and a lower p. I n H, R, K have basic side chains and higher p. I Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 16

Electrophoresis n Proteins have an overall p. I that depends on the net acidity/basicity of the side chains n The differences in p. I can be used for separating proteins on a solid phase permeated with liquid n Different amino acids migrate at different rates, depending on their isoelectric points and on the p. H of the aqueous buffer Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 17

Titration Curves of Amino Acids n If p. Ka values for an amino acid are known the fractions of each protonation state can be calculated (Henderson-Hasselbach Equation) n p. H = p. Ka – log [A-]/[HA] n This permits a titration curve to be calculated or p. Ka to be determined from a titration curve Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 18

26. 3 Synthesis of Amino Acids n Bromination of a carboxylic acid by treatment with Br 2 and PBr 3 (Section 22. 4) then use NH 3 or phthalimide (24. 6) to displace Br Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 19

The Amidomalonate Synthesis n Based on malonic ester synthesis (see 22. 8). n Convert diethyl acetamidomalonate into enolate ion with base, followed by alkylation with a primary alkyl halide n Hydrolysis of the amide protecting group and the esters and decarboxylation yields an -amino Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 20

Reductive Amination of -Keto Acids n Reaction of an -keto acid with NH 3 and a reducing agent (see Section 24. 6) produces an -amino acid Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 21

26. 4 Enantioselective Synthesis of Amino Acids n Amino acids (except glycine) are chiral and pure enantiomers are required for any protein or peptide synthesis n Resolution of racemic mixtures is inherently ineffecient since at least half the material is discarded n An efficient alternative is enantioselective synthesis Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 22

Chemical Resolution of R, S Amino Acids n Convert the amino group into an amide and react with a chiral amine to form diastereomeric salts n Salts are separated and converted back to the amino acid by hydrolysis of the amide Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 23

Enzymic Resolution n Enzymes selectively catalyze the hydrolysis of amides formed from an L

Enantioselective Synthesis of Amino Acids n Chiral reaction catalyst creates diastereomeric transition states that lead to an excess of one enantiomeric product n Hydrogenation of a Z enamido acid with a chiral hydrogenation catalyst produces S enantiomer selectively Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 25

26. 5 Peptides and Proteins n Proteins and peptides are amino acid polymers in which the individual amino acid units, called residues, are linked together by amide bonds, or peptide bonds n An amino group from one residue forms an amide bond with the carboxyl of a second residue Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 26

Peptide Linkages n Two dipeptides can result from reaction between A and S, depending on which COOH reacts with which NH 2 we get AS or SA n The long, repetitive sequence of N CH CO atoms that make up a continuous chain is called the protein’s backbone n Peptides are always written with the N-terminal amino acid (the one with the free NH 2 group) on the left and the C-terminal amino acid (the one with the free CO 2 H group) on the right n Alanylserine is abbreviated Ala-Ser (or A-S), and serylalanine is abbreviated Ser-Ala (or S-A) Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 27

26. 6 Covalent Bonding in Peptides n The amide bond that links different amino acids together in peptides is no different from any other amide bond (see Section 24. 4). Amide nitrogens are nonbasic because their unshared electron pair is delocalized by interaction with the carbonyl group. This overlap of the nitrogen p orbital with the π orbitals of the carbonyl group imparts a certain amount of double-bond character to the C–N bond and restricts rotation around it. The amide bond is therefore planar, and the N–H is oriented 180° to the C=O. Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 28

26. 6 Covalent Bonding in Peptides Based on Mc. Murry, Organic Chemistry, Chapter 26,

Disulfides n Thiols in adjacent chains can form a disulfide RS–SR through spontaneous oxidation (see 18. 10) n A disulfide bond between cysteine residues in different peptide chains links the otherwise separate chains together, while a disulfide bond between cysteine residues in the same chain forms a loop Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 30

26. 7 Structure Determination of Peptides: Amino Acid Analysis n The sequence of amino acids in a pure protein is specified genetically n If a protein is isolated it can be analyzed for its sequence n The composition of amino acids can be obtained by automated chromatography and quantitative measurement of eluted materials using a reaction with ninhydrin that produces an intense purple color Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 31

Amino Acid Analysis Chromatogram Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th

26. 8 Peptide Sequencing: The Edman Degradation n The Edman degradation cleaves amino acids one at a time from the N-terminus and forms a detectable, separable derivative for each amino acid Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 33

26. 9 Peptide Sequencing: C-Terminal Residue Determination n Carboxypeptidase enzymes cleave the C-terminal amide bond n Analysis determines the appearance of the first free amino acid, which must be at the carboxy terminus of the peptide Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 34

26. 10 Peptide Synthesis n Peptide synthesis requires that different amide bonds must be formed in a desired sequence n The growing chain is protected at the carboxyl terminal and added amino acids are N-protected n After peptide bond formation, N-protection is removed Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 35

Carboxyl Protecting Groups n Usually converted into methyl or benzyl esters n Removed by mild hydrolysis with aqueous Na. OH n Benzyl esters are cleaved by catalytic hydrogenolysis of the weak benzylic C–O bond Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 36

Amino Group Protection n An amide that is less stable than the protein amide is formed and then removed n The tert-butoxycarbonyl amide (BOC) protecting group is introduced with di-tert-butyl dicarbonate n Removed by brief treatment with trifluoroacetic acid Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 37

Peptide Coupling n Amides are formed by treating a mixture of an acid and

Overall Steps in Peptide Synthesis Based on Mc. Murry, Organic Chemistry, Chapter 26, 6

26. 11 Automated Peptide Synthesis: The Merrifield Solid-Phase Technique n Peptides are connected to beads of polystyrene, reacted, cycled and cleaved at the end Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 40

Automated Synthesis n The solid-phase technique has been automated, and computer-controlled peptide synthesizers are available for automatically repeating the coupling and deprotection steps with different amino acids Applied Biosystems® Synthesizer Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 41

26. 12 Protein Classification n Simple proteins yield only amino acids on hydrolysis n Conjugated proteins, which are much more common than simple proteins, yield other compounds such as carbohydrates, fats, or nucleic acids in addition to amino acids on hydrolysis. n Fibrous proteins consist of polypeptide chains arranged side by side in long filaments n Globular proteins are coiled into compact, roughly spherical shapes n Most enzymes are globular proteins Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 42

Some Common Fibrous and Globular Proteins Based on Mc. Murry, Organic Chemistry, Chapter 26,

26. 13 Protein Structure n The primary structure of a protein is simply the amino acid sequence. n The secondary structure of a protein describes how segments of the peptide backbone orient into a regular pattern. n The tertiary structure describes how the entire protein molecule coils into an overall three-dimensional shape. n The quaternary structure describes how different protein molecules come together to yield large aggregate structures Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 44

-Keratin n A fibrous structural protein coiled into a right-handed helical secondary structure, -helix stabilized by Hbondsb between amide N–H groups and C=O groups four residues away a-helical segments in their chains Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 45

Fibroin n Fibroin has a secondary structure called a b- pleated sheet in which polypeptide chains line up in a parallel arrangement held together by hydrogen bonds between chains Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 46

Myoglobin n Myoglobin is a small globular protein containing 153 amino acid residues in a single chain n 8 helical segments connected by bends to form a compact, nearly spherical, tertiary structure Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 47

Internal and External Forces n Acidic or basic amino acids with charged side chains congregate on the exterior of the protein where they can be solvated by water n Amino acids with neutral, nonpolar side chains congregate on the hydrocarbon-like interior of a protein molecule n Also important for stabilizing a protein's tertiary structure are the formation of disulfide bridges between cysteine residues, the formation of hydrogen bonds between nearby amino acid residues, and the development of ionic attractions, called salt bridges, between positively and negatively charged sites on various amino acid side chains within the protein Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 48

26. 14 Enzymes n An enzyme is a protein that acts as a catalyst for a biological reaction. n Most enzymes are specific for substrates while enzymes involved in digestion such as papain attack many substrates Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 49

Cofactors n In addition to the protein part, many enzymes also have a nonprotein part called a cofactor n The protein part in such an enzyme is called an apoenzyme, and the combination of apoenzyme plus cofactor is called a holoenzyme. Only holoenzymes have biological activity; neither cofactor nor apoenzyme can catalyze reactions by themselves n A cofactor can be either an inorganic ion or an organic molecule, called a coenzyme n Many coenzymes are derived from vitamins, organic molecules that are dietary requirements for metabolism and/or growth Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 50

Types of Enzymes by Function n Enzymes are usually grouped according to the kind of reaction they catalyze, not by their structures Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 51

26. 15 How Do Enzymes Work? Citrate Synthase n Citrate synthase catalyzes a mixed Claisen condensation of acetyl Co. A and oxaloacetate to give citrate n Normally Claisen condensation require a strong base in an alcohol solvent but citrate synthetase operates in neutral solution Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 52

The Structure of Citrate Synthase n Determined by X-ray crystallography n Enzyme is very large compared to substrates, creating a complete environment for the reaction Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 53

Mechanism of Citrate Synthetase n A cleft with functional groups binds oxaloacetate n Another cleft opens for acetyl Co. A with H 274 and D 375, whose carboxylate abstracts a proton from acetyl Co. A n The enolate (stabilized by a cation) adds to the carbonyl group of oxaloacetate n The thiol ester in citryl Co. A is hydrolyzed Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 54

26. 16 Protein Denaturation n The tertiary structure of a globular protein is the result of many intramolecular attractions that can be disrupted by a change of the environment, causing the protein to become denatured n Solubility is drastically decreased as in heating egg white, where the albumins unfold and coagulate n Enzymes also lose all catalytic activity when denatured Based on Mc. Murry, Organic Chemistry, Chapter 26, 6 th edition, (c) 2003 55