Amino Acids, Pep.des, and Proteins

Transcription

1 Amino Acids, Pep.des, and Proteins 1

2 Proteins Amides from Amino Acids Amino acids contain a basic amino group and an acidic carboxyl group Joined as amides between the NH 2 of one amino acid and the CO 2 H to the next amino acid Chains with fewer than 50 units are called pep.des Protein: large chains that have structural or cataly.c func.ons in biology 2

3 Why this Chapter? Amino acids are the fundamental building blocks of proteins To see how amino acids are incorporated into proteins and the structures of proteins 3

4 Structures of Amino Acids In neutral solu.on, the COOH is ionized and the NH 2 is protonated The resul.ng structures have + and - charges (a dipolar ion, or zwi,erion) They are like ionic salts in solu.on 4

5 The Common Amino Acids 20 amino acids form amides in proteins All are α- amino acids - the amino and carboxyl are connected to the same C They differ by the other subs.tuent awached to the α carbon, called the side chain, with H as the fourth subs.tuent Proline is a five- membered secondary amine, with N and the α C part of a five- membered ring See table 26.1 to examine names, abbrevia.ons, physical proper.es, and structures of 20 commonly occurring amino acids 5

6 Abbrevia.ons and Codes Alanine A, Ala Arginine R, Arg Asparagine N, Asn Aspar.c acid D, Asp Cysteine C, Cys Glutamine Q, Gln Glutamic Acid E, Glu Glycine G, Gly His.dine 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 6

7 Chirality of Amino Acids Glycine, 2- amino- ace.c acid, is achiral In all the others, the α carbons of the amino acids are centers of chirality The stereochemical reference for amino acids is the Fischer projec.on of L- serine Proteins are derived exclusively from L- amino acids 7

8 Types of side chains Neutral: Fibeen of the twenty have neutral side chains Asp and Glu have a second COOH and are acidic Lys, Arg, His have addi.onal basic amino groups side chains (the N in tryptophan is a very weak base) Cys, Ser, Tyr (OH and SH) are weak acids that are good nucleophiles 8

9 His.dine Contains an imidazole ring that is par.ally protonated in neutral solu.on Only the pyridine- like, doubly bonded nitrogen in his.dine is basic. The pyrrole- like singly bonded nitrogen is nonbasic because its lone pair of electrons is part of the 6 π electron aroma.c imidazole ring. 9

10 Essen.al Amino Acids All 20 of the amino acids are necessary for protein synthesis Humans can synthesize only 10 of the 20 The other 10 must be obtained from food 10

11 Amino Acids, the Henderson Hasselbalch Equa.on, and Isoelectric Points In acidic solu.on, the carboxylate and amine are in their conjugate acid forms, an overall ca.on In basic solu.on, the groups are in their base forms, an overall anion In neutral solu.on ca.on and anion forms are present This ph where the overall charge is 0 is the isoelectric point, pi 11

12 Titra.on Curves of Amino Acids If pka values for an amino acid are known the frac.ons of each protona.on state can be calculated (Henderson- Hasselbach Equa.on) ph = pk a log [A - ]/[HA] This permits a.tra.on curve to be calculated or pk a to be determined from a.tra.on curve 12

13 pi Depends on Side Chain The 15 amino acids with thiol, hydroxyl groups or pure hydrocarbon side chains have pi = 5.0 to 6.5 (average of the pk a s) D and E have acidic side chains and a lower pi H, R, K have basic side chains and higher pi 13

14 Electrophoresis Proteins have an overall pi that depends on the net acidity/ basicity of the side chains The differences in pi can be used for separa.ng proteins on a solid phase permeated with liquid Different amino acids migrate at different rates, depending on their isoelectric points and on the ph of the aqueous buffer 14

15 Synthesis of Amino Acids Bromina.on of a carboxylic acid by treatment with Br 2 and PBr 3 then use NH 3 or phthalimide to displace Br 15

16 The Amidomalonate Synthesis Based on malonic ester synthesis. Convert diethyl acetamidomalonate into enolate ion with base, followed by alkyla.on with a primary alkyl halide Hydrolysis of the amide protec.ng group and the esters and decarboxyla.on yields an α- amino acid 16

17 Reduc.ve Amina.on of α- Keto Acids Reac.on of an α- keto acid with NH 3 and a reducing agent produces an α- amino acid 17

18 Enan.oselec.ve Synthesis of Amino Acids Amino acids (except glycine) are chiral and pure enan.omers are required for any protein or pep.de synthesis Resolu.on of racemic mixtures is inherently ineffecient since at least half the material is discarded An efficient alterna.ve is enan.oselec.ve synthesis 18

19 Enan.oselec.ve Synthesis of Amino Acids (cont d) Chiral reac.on catalyst creates diastereomeric transi.on states that lead to an excess of one enan.omeric product Hydrogena.on of a Z enamido acid with a chiral hydrogena.on catalyst produces S enan.omer selec.vely 19

20 Pep.des and Proteins Proteins and pep.des are amino acid polymers in which the individual amino acid units, called residues, are linked together by amide bonds, or pep.de bonds An amino group from one residue forms an amide bond with the carboxyl of a second residue 20

21 Pep.de Linkages Two dipep.des can result from reac.on between A and S, depending on which COOH reacts with which NH 2 we get AS or SA The long, repe..ve sequence of N CH CO atoms that make up a con.nuous chain is called the protein s backbone Pep.des are always wriwen with the N- terminal amino acid (the one with the free NH 2 group) on the leb and the C- terminal amino acid (the one with the free CO 2 H group) on the right Alanylserine is abbreviated Ala- Ser (or A- S), and serylalanine is abbreviated Ser- Ala (or S- A) 21

22 Amino Acid Analysis of Pep.des The sequence of amino acids in a pure protein is specified gene.cally If a protein is isolated it can be analyzed for its sequence The composi.on of amino acids can be obtained by automated chromatography and quan.ta.ve measurement of eluted materials using a reac.on with ninhydrin that produces an intense purple color 22

23 Pep.de Sequencing: The Edman Degrada.on The Edman degrada.on cleaves amino acids one at a.me from the N- terminus and forms a detectable, separable deriva.ve for each amino acid 23

24 Pep.de Synthesis Pep.de synthesis requires that different amide bonds must be formed in a desired sequence The growing chain is protected at the carboxyl terminal and added amino acids are N- protected Aber pep.de bond forma.on, N- protec.on is removed 24

25 Carboxyl Protec.ng Groups Usually converted into methyl or benzyl esters Removed by mild hydrolysis with aqueous NaOH Benzyl esters are cleaved by cataly.c hydrogenolysis of the weak benzylic C O bond 25

26 Amino Group Protec.on An amide that is less stable than the protein amide is formed and then removed The tert- butoxycarbonyl amide (BOC) protec.ng group is introduced with di- tert- butyl dicarbonate Removed by brief treatment with trifluoroace.c acid 26

27 Pep.de Coupling Amides are formed by trea.ng a mixture of an acid and amine with dicyclohexylcarbodiimide (DCC) 27

28 Overall Steps in Pep.de Synthesis 28

29 Automated Pep.de Synthesis: The Merrifield Solid- Phase Technique Pep.des are connected to beads of polystyrene, reacted, cycled and cleaved at the end 29

30 Protein Structure The primary structure of a protein is simply the amino acid sequence. The secondary structure of a protein describes how segments of the pep.de backbone orient into a regular pawern. The terhary structure describes how the en.re protein molecule coils into an overall three- dimensional shape. The quaternary structure describes how different protein molecules come together to yield large aggregate structures 30

31 α- Helix α- Helix stabilized by H- bonds between amide N H groups and C=O groups four residues away 31

32 β- Pleated Sheet β- pleated sheet secondary structure is exhibited by polypep.de chains lined up in a parallel arrangement, and held together by hydrogen bonds between chains 32

33 Denatura.on of Proteins The ter.ary structure of a globular protein is the result of many intramolecular awrac.ons that can be disrupted by a change of the environment, causing the protein to become denatured Solubility is dras.cally decreased as in hea.ng egg white, where the albumins unfold and coagulate Enzymes also lose all cataly.c ac.vity when denatured 33

34 Enzymes and Coenzymes An enzyme is a protein that acts as a catalyst for a biological reac.on. Most enzymes are specific for substrates while enzymes involved in diges.on, such as papain awack many substrates 34

35 Types of Enzymes by Func.on Enzymes are usually grouped according to the kind of reac.on they catalyze, not by their structures 35

36 How Do Enzymes Work? Citrate Synthase Citrate synthase catalyzes a mixed Claisen condensa.on of acetyl CoA and oxaloacetate to give citrate Normally Claisen condensa.ons require a strong base in an alcohol solvent but citrate synthetase operates in neutral solu.on 36

37 The Structure of Citrate Synthase Determined by X- ray crystallography Enzyme is very large compared to substrates, crea.ng a complete environment for the reac.on 37

38 Mechanism of Citrate Synthetase A cleb with func.onal groups binds oxaloacetate Another cleb opens for acetyl CoA with H 274 and D 375, which have carboxylate that abstract a proton from acetyl CoA The enolate (stabilized by a ca.on) adds to the carbonyl group of oxaloacetate The thiol ester in citryl CoA is hydrolyzed 38