Chapter 27: Amino Acids, Peptides, and Proteins. monomer unit: αamino acids 2 C 2! Amino Acid = sidechain Biopolymer: the monomeric amino acids are linked through an amide bond (the carboxylic acids of one AA with the αamino group of a second) 1 1 2 2 C 3 C 2 3 C 3 2 2 terminus 2 Cterminus 1 2 3 4 5 6 7 Peptide or protein (polypeptide) peptide (< 50 amino acids) protein (> 50 amino acids) 307 27.1: Classification of Amino Acids. AA s are classified according to the location of the amino group. 2 C C 2!amino acid (2amino carboxylic acid) 2 C C C 2 "amino acid (3amino carboxylic acid) 2 C C C C 2 #amino acid (4amino carboxylic acid) There are 20 genetically encoded αamino acids found in peptides and proteins 19 are primary amines, 1 (proline) is a secondary amine 19 are chiral, 1 (glycine) is achiral; the natural configuration of the αcarbon is L. C C 2 Dglyceraldehyde C C 2 Lglyceraldehyde C 2 2 C 3 Lalanine C 2 2 C C 2 Derythrose C C 2 Lerythrose C 2 2 C 3 Ltheronine (2,3) C 2 2 3 C C 2 C 3 Lisoleucine (2,3) 308 157
αamino acids are classified by the properties of their sidechains. onpolar: C C C 3 Glycine (Gly, G) 3 ()()Alanine (Ala, A) 3 ()()Valine (Val, V) C C C 3 ()( )Leucine (Leu, L) 3 (2,3)()Isoleucine (Ile, I) 3 ()( )Methionine (Met, M) C 3 C 3 C ()( )Proline (Pro, P) ()( )enylalanine (e, F) ()( )Tryptophan (Trp, W) Polar but nonionizable: C C C 3 3 3 ()( )erine (er, ) (2,3)( )Threonine (Thr, T) ()( )Tyrosine (Tyr, Y) pka ~ 13 pka ~ 13 pka ~ 10.1 C C 2 3 3 ()( )Cysteine (Cys, C) ()( )Asparagine (Asn, ) pka ~ 8.2 C 2 3 ()()Glutamine (Gln, Q) 309 Acidic: C C 3 3 ()()Aspartic Acid (Asp, D) ()()Glutamic Acid (Glu, E) pka ~ 3.6 pka ~ 4.2 Basic: 3 C 3 C C 3 2 3 ()()Lysine (Lys, K) ()( )istidine (is, ) ()()Arginine (Arg, ) pka ~ 10.5 pka ~ 6.0 pka ~ 12.5 27.2: tereochemistry of Amino Acids: The natural configuration of the αcarbon is L. DAmino acids are found in the cell walls of bacteria. The Damino acids are not genetically encoded, but derived from the epimerization of Lisomers 310 158
27.3: AcidBase Behavior of Amino Acids. Amino acids exist as a zwitterion: a dipolar ion having both a formal positive and formal negative charge (overall charge neutral). _ 2 C 2 3 C 2 pk a ~ 5 pk a ~ 9 Amino acids are amphoteric: they can react as either an acid or a base. Ammonium ion acts as an acid, the carboxylate as a base. Isoelectric point (pi): The p at which the amino acid exists largely in a neutral, zwitterionic form (influenced by the nature of the sidechain) 3 C 2 low p 3 pka 1 3 _ C 2 _ pka 2 _ 2 C 2 high p Table 27.2 (p. 1115) & 27.2 (p. 1116) 311 pi = pka x pka y 2 C 3 3 C 2 3 pka 1 (2.3) low p C 3 C 2 pka 2 (9.7) C 3 2 C 2 high p C 2 C 2 C 2 C 2 C 2 3 C 2 low p pka 1 (1.9) 3 C 2 C 2 pka 3 (3.6) 3 C 2 C 2 pka 2 (9.6) C 2 2 C 2 high p 3 3 3 2 (C 2 ) 4 3 C 2 low p pka 1 (2.2) (C 2 ) 4 3 C 2 pka 2 (9.0) (C 2 ) 4 2 C 2 pka 3 (10.5) (C 2 ) 4 2 C 2 high p 312 159
Electrophoresis: separation of polar compounds based on their mobility through a solid support. The separation is based on charge (pi) or molecular mass. 313 27.5: ynthesis of Amino Acids: Br Br 2, PBr 3 3 C 2 C 2 C C 2 Ch. 19.16 trecker ynthesis: recall reductive amination 2 C C 2 3 2 C C 2 C C 2 ab(c) 3 2 C C 2 3 C 2 C ac! 2 C C! 3 or a, 2 2 C C 2!C: Amidomalonate ynthesis: recall the malonic acid synthesis C 2 Et Et a C C 2 Et C 2 X C 2 Et C C 2 C 2 Et 3 2 C 2 C 2 C C 2 314 160
27.5: eactions of Amino Acids. Amino acids will undergo reactions characteristic of the amino (amide formation) and carboxylic acid (ester formation) groups. 2 C 2 C 2 C 3 C 2 C 3 3 3 C C 3 C 2 3 C base C 2 27.6: ome Biochemical eactions of Amino Acids. Many enzymes involved in amino acid biosynthesis, metabolism and catabolism are pyridoxal phosphate (vitamin B 6 ) dependent (please read) C 2 3 Lamino acid 2 3 P pyridoxal phosphate (PLP) P transaminase C 2 C 2 racemase, epimerase C 2 3 Damino acid decarboxylase 3 315 27.7: Peptides. Proteins and peptides are polymers made up of amino acid units (residues) that are linked together through the formation of amide bonds (peptide bonds) from the amino group of one residue and the carboxylate of a second residue 2 C 2 Alanine 2 2 C 2 2 erine terminus C 2 Cterminus 2 Ala er (A ) terminus 2 er Ala ( A) Cterminus C 2 By convention, peptide sequences are written left to right from the terminus to the Cterminus 1 2 3 4 5 6 7 backbone 316 161
The amide (peptide) bond has C= double bond character due to resonance resulting in a planar geometry 2 1 amide bond _ 2 1 restricts rotations resistant to hydrolysis The bond of one amide linkage can form a hydrogen bond with the C= of another. distance 2.85 3.20 Å optimal angle is 180 Disulfide bonds: the thiol groups of cysteine can be oxidized to form disulfides (CysCys) 2 2 2 C 1/2 2 2 2 2 2 C 2 C 2 317 1 2 6 4 8 5 10 9 1/2 2 2 1 2 9 4 11 5 12 13 Epidermal Growth Factor (EGF): the miracle of mother s spit 53 amino acid, 3 disulfide linkages 1986 obel Prize in Medicine: tanley Cohen ita LeviMontalcini 318 162
27.8: Introduction to Peptide tructure Determination. Protein tructure: primary (1 ) structure: the amino acid sequence secondary (2 ): frequently occurring substructures or folds tertiary (3 ): threedimensional arrangement of all atoms in a single polypeptide chain quaternary (4 ): overall organization of noncovalently linked subunits of a functional protein. 1. Determine the amino acids present and their relative ratios 2. Cleave the peptide or protein into smaller peptide fragments and determine their sequences 3. Cleave the peptide or protein by another method and determine their sequences. Align the sequences of the peptide fragments from the two methods 319 EAYLVCGE FVQLFLK GCFLPK LGA FVQLF LKEAY LVCGEGCF LPKLGA FVQLF FVQLFLK LKEAY EAYLVCGE LVCGEGCF GCFLPK LPKLGA LGA FVQLFLKEAYLVCGEGCFLPKLGA 320 163
27.9: Amino Acid Analysis. automated method to determine the amino acid content of a peptide or protein eaction of primary amines with ninhydrin 3 C 2 [] inhydrin reduce any disulfide bonds Enzymatic digestion peptide orprotein or 3, Δ 3 C 2 C C 2 individual amino acids liquid chromatography derivatize w/ ninhydrin Detected w/ UVvis Different amino acids have different chromatographic mobilities (retention times) 1972 obel Prize in Chemistry William tein tanford Moore 321 27.10: Partial ydrolysis of Peptides. Acidic hydrolysis of peptides cleave the amide bonds indiscriminately. Proteases (peptidases): Enzymes that catalyzed the hydrolysis of the amide bonds of peptides and proteins. Enzymatic cleavage of peptides and proteins at defined sites: trypsin: cleaves at the Cterminal side of basic residues, Arg, Lys but not is 3 1 3 C 2 trypsin 2 3 1 3 3 C 2 3 chymotrypsin: cleaves at the Cterminal side of aromatic residues e, Tyr, Trp 3 3 1 3 C 2 chymotrypsin 2 3 1 3 3 C 2 322 164
Trypsin and chymotrypsin are endopeptidases Carboxypeptidase: Cleaves the amide bond of the Cterminal amino acid (exopeptidase) 27.11: End Group Analysis. The Cterminal AA is identified by treating with peptide with carboxypeptidase, then analyzing by liquid chormatography (AA Analysis). labeling: The peptide is first treated with 1fluoro2,4dinitro benzene (anger s reagent), which selectively reacts with the terminal amino group. The peptide is then hydrolyzed to their amino acids and the terminal amino acid identified as its (2,4dinitrophenyl) derivative (DP). 2 2 F enzymatic digestion or 3,! 2 3 1 C2 2 1 2! nucleophilic aromatic substitution 2 2 plus other unlabeled amino acids 1 C2 323 27.12: Insulin. (please read) Insulin has two peptide chains (the A chain has 21 amino acids and the B chain has 30 amino acids) held together by two disulfide linkages Pepsin: cleaves at the Cterminal side of e, Tyr, Leu; but not at Val or Ala Pepsin cleavage Trypsin cleavage 3 cleavage 324 165
27.13: The Edman Degradation and Automated Peptide equencing. Chemical method for the sequential cleavage and identification of the amino acids of a peptide, one at a time starting from the terminus. eagent: =C=, phenylisothiocyanate (PITC) C 2 1 1 C2 p 9.0 C 2 1 1 C2 2 C 2 1 peptide with a new terminal amino acid (repeat degradation cycle) phenylthiohydantoin: separated by liquid chromatography (based of the group) and detected by UVvis 1 325 Peptide sequencing by Edman degradation: Cycle the p to control the cleavage of the terminal amino acid by PITC. Monitor the appearance of the new phenylthiohydantoin for each cycle. Good for peptides up to ~ 25 amino acids long. Longer peptides and proteins must be cut into smaller fragments before Edman sequencing. Tandem mass spectrometry has largely replaced Edman degradation for peptide sequencing 27.14: The trategy for Peptide ynthesis: Chemical synthesis of peptide: 1. olution phase synthesis 2. olidphase synthesis 326 166
2 Val Ala (V A) C 2 2 2 Ala C 2 2 Val C 2 2 2 Ala Val (A V) C 2 The need for protecting groups P n 2 P c peptide coupling 2 P n P c selectively remove P n Ala Val Ala Val (A V) 2 Ala Val (A V) P c peptide coupling ( 2 ) P n e (F) P n rthogonal protecting group strategy: the carboxylate protecting group must be stable to the reaction conditions for the removal of the αamino protecting group and ( vice versa) e Ala Val (F A V) P c epeat peptide synthesis 327 27.15: Amino Group Protection. The αamino group is protected as a carbamate. tertbutoxycarbonyl (tbc) removed with mild acid 3 C 6 5 Cl 27.16: Carboxyl Group Protection. Protected as a benzyl ester; removed by hydrogenolysis 2 Base benzyloxycarbonyl (cbz) removed with mild acid or by hydrogenolysis fluorenylmethylcarbonyl (FMC) removed with mild base (piperidine) peptide coupling C C 6 2 6 5 5 C 6 5 C 6 5 2 C C 6 6 5 5 2, Pd/C C 6 5 C 6 5 peptide 2 coupling mild acid 3 328 167
27.17: Peptide Bond Formation. Amide formation from the reaction of an amine with a carboxylic acid is slow. Amide bond formation (peptide coupling) can be accelerated if the carboxylic acid is activated. eagent: dicyclohexylcarbodiimide (DCC) C 6 11 C C 6 11 C 6 11 C C 6 11 (DCC) C 6 11 C ' C6 11 ' 2 Amide C 6 11 C ' C 6 11 "activated acid" ' C 6 11 C 6 11 DCU C 6 11 C C 6 11 cbz Ala Bn 2 Val DCC peptide coupling cbz Bn CF 3 C 2 selectively remove protecting group 2 Bn DCC cbz e (F) cbz Bn 2, Pd/C 2 e Ala Val (F A V) 329 In order to practically synthesize peptides and proteins, time consuming purifications steps must be avoided until the very end of the synthesis. Large excesses of reagents are used to drive reactions forward and accelerate the rate of reactions. ow are the excess reagents and byproducts from the reaction, which will interfere with subsequent coupling steps, removed without a purification step? 27.18: olidase Peptide ynthesis: The Merrifield Method. Peptides and proteins up to ~ 100 residues long are synthesized on a solid, insoluble, polymer support. Purification is conveniently accomplished after each step by a simple wash and filtration. 330 168
The solid support (Merrifield resin): polystyrene polymer styrene initiator polymerization 3 CC 2 Cl ZnCl 2 C 2 Cl divinylbenzene (crosslinker, ~1 %) _ BC olidphase peptide synthesis BC CF 3C 2 2 2 Val FMC DCC FMC peptide coupling purify: wash & filter remove protecting group 2 purify: wash & filter DCC FMC e (F) FMC purify: wash & filter remove protecting group F remove protecting group and cleave from solidsupport purify by liquid chromatograrphy or electrophoresis 2 331 ibonuclease A 124 amino acids, catalyzes the hydrolysis of A olidphase synthesis of ase A: ynthetic ase A: 78 % activity 0.4 mg was synthesized 2.9 % overall yield average yield ~ 97% per coupling step is119 A is12 A LY GLU T ALA ALA ALA LY PE GLU AG GL I MET AP E E T E ALA ALA E E E A TY CY A GL MET MET LY E AG A LEU T LY AP AG CY LY P VAL A T PE VAL I GLU E LEU ALA AP VAL GL ALA VAL CY E GL LY A VAL ALA CY LY A GLY GL T A CY TY GL E TY E T MET E ILE T AP CY AG GLU T GLY E E LY TY P A CY ALA TY LY T T GL ALA A LY I ILE ILE VAL ALA CY GLU GLY A P TY VAL P VAL I PE AP ALA E VAL is119 B is12 B pdb code: 1AFL. Bruce Merrifield, ockefeller University, 1984 obel Prize in Chemistry: for his development of methodology for chemical synthesis on a solid matrix. 332 169
170 333 27.19: econdary tructures of Peptides and Proteins. βsheet: Two or more extended peptide chain, in which the amide backbones are associated by hydrogen bonded parallel crossover C C C C antiparallel loop or turn C C C C 334 αhelix: 3.6 amino acids per coil, 5.4 Å 3.6 AA 5.4 Å C
myoglobin pdb code: 1WLA Bacteriorhodopsin pdb code: 1AP9 Parallel βsheets carbonic anhydrase pdb code: 1QM Antiparallel βsheets of lectin pdb code: 2LAL 335 27.20: Tertiary tructure of polypeptides and Proteins. Fibrous. Polypeptides strands that bundle to form elongated fibrous assemblies; insoluble; Globular. Proteins that fold into a spherical conformation. ydrophobic effect. Proteins will fold so that hydrophobic amino acids are on the inside (shielded from water) and hydrophilic amino acids are on the outside (exposed to water). Pro Ile Lys Tyr Leu Glu e Ile er Asp Ala Ile Ile is Val is er Lys 336 171
Enzymes: proteins that catalyze biochemical reactions. by bringing the reactive atoms together in the optimal geometry for the reaction. lowering the activation energy (ΔG ) by stabilizing the transition state and/or high energy intermediate. many enzymes use the functional groups of the amino acid sidechain to carry out the reactions Proteases (peptidases): catalyzes the hydrolysis of peptide bonds 3 C 2 protease 2 3 3 Four classes of proteases: erine (trypsin): aspartatehistidineserine Aspartyl (IV protease, renin): two aspartates Cysteine (papain, caspase): histidinecysteine Metallo (Zn 2 ) (carboxypeptidase, ACE): glutamate C 2 337 Mechanism of carboxylpeptidase, metalloprotease (p. 1151) Mechanism of a serine protease (trypsin, chymotrypsin): er 195 is 57 oxyanion hole 1 2 1 2 er 195 is 57 2 2 is 57 er 192 1 acylenzyme intermediate Asp 102 C 2 Asp 102 C 2 Asp 102 C 2 er 192 1 is 57 er 195 is 57 C 2 Asp 102 C 2 Asp 102 C 2 338 172
27.21: Coenzymes. ome reactions require additional organic molecules or metal ions. These are referred to as cofactors or coenzymes. Fe eme Biotin (vitamin B 7 ) 2 C 2 P P Thiamin Diphosphate (vitamin B 1 ) 2 Folic Acid (vitamin B 9 ) P Pyridoxal ophates (vitamin B 6 ) P C 2 P C 2 Flavin Adenine Diphosphate (FAD) (Vitamin B 2 ) 2 2 2 2 C Co P Vitamin B 12 (cyanocobalamin) 2 2 2 27.22: Protein Quaternary tructure. (please read) 339 173