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18 Introduction to Organic Chemistry 2 ed William H. Brown 18-1 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Amino Acids & Proteins Chapter 18 18-2 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Amino Acids • Amino acid: a compound that contains both an amino group and a carboxyl group • a-Amino acid: an amino acid in which the amino group is on the carbon adjacent to the carboxyl group • although a-amino acids are commonly written in the unionized form, they are more properly written in the zwitterion (internal salt) form R- CH- CO2 H N H2 unionized form R- CH- CO2 N H3 + zwitterion 18-3 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Chirality of Amino Acids • With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the acarbon) and are chiral • the vast majority of a-amino acids have the L-configuration at the a -carbon CO 2 H NH 3 + CH 3 D-Alanine CO 2 + H3 N H CH 3 L-Alanine 18-4 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 20 Protein-Derived AA Nonpolar side chains (predominant form at pH 7.0) glycine (gly, G) Halanine (ala, A) CH3 valine (val, V) ( CH3 ) 2 CH leucine (leu, L) ( CH3 ) 2 CHCH 2 isoleucine (ile, I) CH3 CH2 CH( CH3 ) methionine (met, M) CH3 S CH2 CH2 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. phenylalanine (phe,F) tryptophan (trp, W) N H proline (Pro, P) + N H H 18-5 18 20 Protein-Derived AA Polar side chains (predominant form at pH 7.0) asparagine (asn, N) O H2 NCCH 2 - serine (ser, S) glutamine (glu, G) O H2 NCCH 2 CH2 - threonine (thr, T) OH CH3 CH- HOCH2 - 18-6 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 20 Protein-Derived AA Acidic side chains (predominant form at pH 7.0) aspartic acid (asp, D) - O 2 CCH2 - glutamic acid (glu, E) - O 2 CCH2 CH2 - cysteine (cys, C) HS CH 2 tyrosine (tyr, Y) HO CH2 - 18-7 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 20 Protein-Derived AA Basic side chains (predominant form at pH 7.0) arginine (arg, R) NH2 + H2 NCNHCH2 CH2 CH2 lysine (lys, K) histidine (his, H) N N H CH2 - + H3 NCH2 CH2 CH2 CH2 - 18-8 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 20 Protein-Derived AA • Structural features • all 20 are a-amino acids • for 19 of the 20, the a-amino group is primary; for proline it is secondary • with the exception of glycine, the a-carbon of each is a stereocenter • isoleucine and threonine contain a second stereocenter • the sulfhydryl group of cysteine, the imidazole group of histidine, and the phenolic hydroxyl of phenylalanine are partially ionized at pH 7.0, but the ionic form is not the major form at this pH 18-9 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 AcidBase Properties Nonpolar & polar side chains alanine asparagine glutamine glycine isoleucine leucine methionine phenylalanine proline serine threonine tryptophan valine Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. pK a of pK a of aCO 2 H aNH 3 2.35 2.02 2.17 2.35 2.32 2.33 2.28 2.58 2.00 2.21 2.09 2.38 2.29 9.87 8.80 9.13 9.78 9.76 9.74 9.21 9.24 10.60 9.15 9.10 9.39 9.72 + 18-10 18 Acid-Base Properties Acidic Side Chains aspartic acid glutamic acid cysteine tyrosine pK a of Basic Side Chains arginine histidine lysine pK a of pK a of aCO 2 H aNH 3 2.10 9.82 2.10 9.47 2.05 10.25 2.20 9.11 pK a of aCO 2 H aNH 3 2.01 9.04 1.77 9.18 2.18 8.95 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. + + pK a of Side Chain 3.86 4.07 8.00 10.07 Side Chain Group carboxyl carboxyl sufhydryl phenolic pK a of Side Chain 12.48 6.10 10.53 Side Chain Group guanidino imidazole 1° amino 18-11 18 Acidity: a-CO2H Groups • The average pKa of an a-carboxyl group is 2.19, which makes it a considerably stronger acid than acetic acid (pKa 4.76) • the greater acid strength is due to the electronwithdrawing inductive effect of the -NH3+ group The ammonium ion has an electron-withdrawing inductive effect RCHCO 2 H + H2 O N H3 + pK a = 2.19 RCHCO 2 NH 3 - + H3 O + + 18-12 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Acidity: side chain -CO2H • Side chain -CO2H groups are stronger acids than acetic acid • the greater acid strength is due to the electronwithdrawing inductive effect of the a-NH3+ group, • the effect decreases with distance from the a-NH3+ group pK a 2.35 CH3 CHCO 2 H pK a 3.86 HO 2 CCH 2 CHCO2 - N H3 + N H3 + pK a 4.07 HO 2 CCH2 CH 2 CHCO2 N H3 + 18-13 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Acidity: a-NH3+ groups • The average value of pKa for an a-NH3+ group is 9.47, compared with an average value of 10.60 for a 1° alkylammonium ion RCHCO 2 NH 3 - + H2 O pK a = 9.47 + NH 2 CH3 CHCH 3 + H2 O NH3 + RCHCO2 + H3 O pK a = 10.76 + CH3 CHCH 3 + H3 O + NH2 18-14 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Basicity-Guanidine Group • The side chain of arginine is a considerably stronger base than an aliphatic amine • its basicity is due to the large resonance stabilization of the protonated form relative to the neutral form •• •• NH2 NH2 •• RNH C •• + NH2 + RNH C RNH C •• + •• NH2 NH2 NH2 •• H2 O NH •• + H3 O RNH C •• NH2 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. + pKa = 12.48 18-15 18 Basicity- Imidazole Group • The imidazole group on the side chain of histidine is a heterocyclic aromatic amine H + H N N N H CH2 CHCO 2 + N H3 - N this lone pair is not a part of the N aromatic sextet; it is H the proton acceptor N+ H CH2 CHCO 2 + NH 3 CH2 CHCO 2 + N H3 - + H3 O - + H2 O pK a 6.10 18-16 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Isoelectric Point • Isoelectric point, pI: the pH at which the majority of amino acid molecules in solution have no net charge • the pI for glycine, for example, falls between the pKa values for the carboxyl and amino groups pI = 1 + 2 (pK a a CO2 H + pK a a NH3 ) = 1 2 (2.35 + 9.78) = 6.06 • given in the following tables are isoelectric points for the 20 protein-derived amino acids 18-17 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Nonpolar & polar side chains alanine asparagine glutamine glycine isoleucine leucine methionine phenylalanine proline serine threonine tryptophan valine pKa of Isoelectric + Side aCO2 H aNH3 Chain Point (pI) ---2.35 9.87 6.11 ---2.02 8.80 5.41 2.17 9.13 ---5.65 2.35 9.78 ---6.06 2.32 9.76 ---6.04 2.33 9.74 ---6.04 2.28 9.21 ---5.74 2.58 9.24 ---5.91 ---2.00 10.60 6.30 2.21 9.15 5.68 ---2.09 9.10 ---5.60 ---2.38 9.39 5.88 2.29 9.72 ---6.00 18-18 pKa of Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. pKa of 18 Acidic Side Chains aspartic acid glutamic acid cysteine tyrosine Basic Side Chains arginine histidine lysine pKa of Isoelectric + Side aCO2 H aNH 3 Chain Point (pI) 2.10 9.82 3.86 2.98 2.10 9.47 4.07 3.08 2.05 10.25 8.00 5.02 2.20 9.11 10.07 5.63 pKa of pKa of pKa of Isoelectric + Side aCO2 H aNH 3 Chain Point (pI) 2.01 9.04 12.48 10.76 1.77 9.18 6.10 7.64 2.18 8.95 10.53 9.74 pKa of Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. pKa of 18-19 18 Electrophoresis • Electrophoresis: the process of separating compounds on the basis of their electric charge • electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose acetate as solid supports • In paper electrophoresis • a paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between two electrode vessels 18-20 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Electrophoresis • a sample of amino acids is applied as a spot on the paper strip • an electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own • molecules with a high charge density move faster than those with low charge density • molecules at their isoelectric point remain at the origin • after separation is complete, the strip is dried and developed to make the separated amino acids visible 18-21 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Electrophoresis • a reagent commonly used to detect amino acid is ninhydrin O O OH RCHCO - + 2 OH + NH3 An a-amino acid O Ninhydrin O O - N O O Purple-colored anion Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. O + RCH + CO 2 + H3 O + 18-22 18 Polypeptides & Proteins • In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds to which he gave the name peptide bonds • Peptide bond: the special name given to the amide bond between the a-carboxyl group of one amino acid and the a-amino group of another 18-23 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Serylalanine (Ser-Ala) 18-24 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Peptides • peptide: the name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain • dipeptide: a molecule containing two amino acids joined by a peptide bond • tripeptide: a molecule containing three amino acids joined by peptide bonds • polypeptide: a macromolecule containing many amino acids joined by peptide bonds • protein: a biological macromolecule of molecular weight 5000 g/mol of greater, consisting of one or more polypeptide chains 18-25 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Writing Peptides • by convention, peptides are written from the left, beginning with the free -NH3+ group and ending at the right with the free -CO2- group peptide bonds C-terminal N-terminal amino acid amino acid O O + H3 NCHC- NHCHC- NHCHCO 2 CH2 OH CH2 C6 H5 CH2 CO 2 - Ser-Phe-Asp 18-26 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Ser-Phe-Asp 18-27 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Primary Structure • Primary structure: the sequence of amino acids in a polypeptide chain, read from the N-terminal amino acid to the C-terminal amino acid • Amino acid analysis • hydrolysis of the polypeptide, most commonly carried out using 6 M HCl at elevated temperature • quantitative analysis of the hydrolysate by ionexchange chromatography 18-28 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Cyanogen Bromide • Cyanogen bromide, BrCN, is specific for cleavage of peptide bonds formed by the carboxyl group of methionine cyanogen bromide is specific for the cleavage of this peptide bond from the N-terminal end O O P N -C- NH CH C NH- P C from the C-terminal end CH2 CH2 -S -CH3 18-29 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Cyanogen Bromide O O P N -C- NH CH C NH- P C + Br C N 0.1M HCl H2 O Cyanogen CH2 bromide CH2 -S -CH3 from the N-terminal end O from the C-terminal end O C + H2 N- PC + CH3 -S -C N Methyl CH2 CH2 thiocyanate (A substituted -lactone) 18-30 P N -C- N H CH Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. O 18 Enzyme Catalysis • A group of protein-cleaving enzymes can be used to catalyze the hydrolysis of specific peptide bonds. Among them are: Enzyme Catalyzes Hydrolysis of Peptide Bond Formed by Carboxyl Group of trypsin chymotrypsin arginine, lysine phenylalanine, tryptophan, tyrosine 18-31 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Edman Degradation • Edman degradation: cleaves the N-terminal amino acid of a polypeptide chain R O H2 N- CH- C- NH- PC + Ph - N= C= S Phenyl isothiocyanate R CH C O + H2 N- P C HN C N S Ph A phenylthiohydantoin 18-32 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Primary Structure • Example 18.6 Deduce the 1° structure of this pentapeptide Experimental Procedure Pentapeptide Edman Degradation Hydrolysis - Chymotrypsin Fragment A Fragment B Hydrolysis - Trypsin Fragment C Fragment D Amino Acid Composition Arg, Glu, His, Phe, Ser Glu Glu, His, Phe Arg, Ser Arg, Glu, His, Phe Ser 18-33 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Peptide Bond Geometry • The four atoms of a peptide bond and the two alpha carbons bonded to it lie in a plane with bond angles of approximately 120° about C and N 18-34 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Peptide Bond Geometry • to account for this geometry, Linus Pauling proposed that a peptide bond is most accurately represented as a hybrid of two contributing structures • the hybrid has considerable C-N double bond character and rotation about a peptide bond is restricted Ca •• C O + C N Ca •• •• O •• •• •• H (1) Ca N Ca H (2) 18-35 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Peptide Bond Geometry • two conformations are possible for a planar peptide bond • virtually all peptide bonds in naturally occurring proteins studied to date have the s-trans conformation O Ca •• •• •• •• H O •• C Ca •• N C H s-trans N Ca Ca s-cis 18-36 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Secondary Structure • Secondary structure: the ordered arrangements (conformations) of amino acids in localized regions of a polypeptide or protein • To determine from model building which conformations would be of greatest stability, Pauling and Corey assumed that • all six atoms of each peptide bond lie in the same plane and in the s-trans conformation • there is hydrogen bonding between the N-H group of one peptide bond and a C=O group of another peptide bond as shown in the next screen 18-37 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Secondary Structure 18-38 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Secondary Structure • On the basis of model building, Pauling and Corey proposed that two types of secondary structure should be particularly stable • the a-helix • the antiparallel b-pleated sheet • a-Helix: a type of secondary structure in which a section of polypeptide chain coils into a spiral, most commonly a right-handed spiral 18-39 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 The a-Helix • polyalanine, cylindrical bonds model, viewed along its length 18-40 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 The a-Helix • polyalanine, cylindrical bonds model, viewed from one end 18-41 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 The a-Helix • polyalanine, space filling model, viewed along its length 18-42 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 The a-Helix • polyalanine, space filling model, viewed from one end 18-43 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 The a-Helix • Structural features of the a-helix • there are 3.6 amino acids per turn of the helix • each peptide bond is s-trans and planar • N-H groups of all peptide bonds point in the same direction, which is roughly parallel to the axis of the helix • C=O groups of all peptide bonds point in the opposite direction, and also parallel to the axis of the helix • the C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it • all R- groups point outward from the helix 18-44 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 b-Pleated Sheet • The antiparallel b-pleated sheet consists of adjacent polypeptide chains running in opposite directions • each peptide bond is planar and has the s-trans conformation • the C=O and N-H groups of peptide bonds from adjacent chains point toward each other and are in the same plane so that hydrogen bonding is possible between them • all R- groups on any one chain alternate, first above, then below the plane of the sheet, etc. 18-45 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 b-Pleated Sheet • polyalanine 18-46 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Tertiary Structure • Tertiary structure: the three-dimensional arrangement in space of all atoms in a single polypeptide chain • disulfide bonds between the side chains of cysteine play an important role in maintaining 3° structure H O H O N N N H CH2 CH2 SH oxidation CH2 CH2 S CH2 CH2 SH reduction CH2 CH2 S O Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. N H O 18-47 18 Quaternary Structure • Quaternary structure: the arrangement of polypeptide chains into a noncovalently bonded aggregation • the major factor stabilizing the aggregation of polypeptide subunits is the hydrophobic effect • Hydrophobic effect: the tendency of nonpolar groups to cluster together in such a way as to be shielded from contact with an aqueous environment 18-48 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Quaternary Structure • if two polypeptide chains, for example, each have one hydrophobic patch, each patch can be shielded from contact with water if the chains form a dimer Protein Number of Subunits alcohol dehydrogenase aldolase hemoglobin lactate dehydrogenase insulin glutamine synthetase tobacco mosaic virus protein disc 2 4 4 4 6 12 17 18-49 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved. 18 Amino Acids & Proteins End of Chapter 18 18-50 Copyright © 2000 by John Wiley & Sons, Inc. All rights reserved.