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Chapter 25 Amino Acids, Peptides, and Proteins 25.1 Classification of Amino Acids Fundamentals While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively. They are classified as , , , etc. amino acids according the carbon that bears the nitrogen. Amino Acids + NH3 CO2– + – H3NCH2CH2CO2 + – H3NCH2CH2CH2CO2 an -amino acid that is an intermediate in the biosynthesis of ethylene a -amino acid that is one of the structural units present in coenzyme A a -amino acid involved in the transmission of nerve impulses The 20 Key Amino Acids More than 700 amino acids occur naturally, but 20 of them are especially important. These 20 amino acids are the building blocks of proteins. All are -amino acids. They differ in respect to the group attached to the carbon. These 20 are listed in Table 25.1. Table 25.1 H + H3N C O C O – R The amino acids obtained by hydrolysis of proteins differ in respect to R (the side chain). The properties of the amino acid vary as the structure of R varies. Table 25.1 H + H3N C O C O – R The major differences among the side chains concern: Size and shape Electronic characteristics Table 25.1 General categories of -amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Table 25.1 H Glycine (Gly or G) + H3N C O C O – H Glycine is the simplest amino acid. It is the only one in the table that is achiral. In all of the other amino acids in the table the carbon is a chirality center. Table 25.1 H + H3N C O C O – CH3 Alanine (Ala or A) Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar and hydrophobic. Table 25.1 H + H3N C O C O CH(CH3)2 Valine (Val or V) – Table 25.1 H + H3N C O C O – CH2CH(CH3)2 Leucine (Leu or L) Table 25.1 H + H3N C O C O – CH3CHCH2CH3 Isoleucine (Ile or I) Table 25.1 H + H3N C O C O – CH3SCH2CH2 Methionine (Met or M) The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat polarizable. Table 25.1 H + H2N C O C CH2 H2C C H2 O – Proline (Pro or P) Proline is the only amino acid that contains a secondary amine function. Its side chain is nonpolar and cyclic. Table 25.1 H + H3N C CH2 O C O – Phenylalanine (Phe or F) The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group. Table 25.1 H + H3N C O C O – Tryptophan CH2 (Trp or W) N H The side chain in tryptophan (a nonpolar amino acid) is larger and more polarizable than the benzyl group of phenylalanine. Table 25.1 General categories of -amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Table 25.1 H + H3N C O C O – CH2OH Serine (Ser or S) The —CH2OH side chain in serine can be involved in hydrogen bonding. Table 25.1 H + H3N C O C O – CH3CHOH Threonine (Thr or T) The side chain in threonine can be involved in hydrogen bonding, but is somewhat more crowded than in serine. Table 25.1 H + H3N C O C O – CH2SH Cysteine (Cys or C) The side chains of two remote cysteines can be joined by forming a covalent S—S bond. Table 25.1 H + H3N C O C O – Tyrosine (Tyr or Y) CH2 OH The side chain of tyrosine is similar to that of phenylalanine but can participate in hydrogen bonding. Table 25.1 H + H3N C H2NCCH2 O C O – Asparagine (Asn or N) O The side chains of asparagine and glutamine (next slide) terminate in amide functions that are polar and can engage in hydrogen bonding. Table 25.1 H + H3N C H2NCCH2CH2 O O C O – Glutamine (Gln or Q) Table 25.1 General categories of -amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Table 25.1 H + H3N – C OCCH2 O C O – Aspartic Acid (Asp or D) O Aspartic acid and glutamic acid (next slide) exist as their conjugate bases at biological pH. They are negatively charged and can form ionic bonds with positively charged species. Table 25.1 H + H3N – C OCCH2CH2 O C O – Glutamic Acid (Glu or E) O Table 25.1 General categories of -amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Table 25.1 H Lysine (Lys or K) + H3N C O C O – + CH2CH2CH2CH2NH3 Lysine and arginine (next slide) exist as their conjugate acids at biological pH. They are positively charged and can form ionic bonds with negatively charged species. Table 25.1 H Arginine (Arg or R) + H3N C O C O – CH2CH2CH2NHCNH2 + NH2 Table 25.1 H Histidine + H3N (His or H) C CH2 N NH O C O – Histidine is a basic amino acid, but less basic than lysine and arginine. Histidine can interact with metal ions and can help move protons from one site to another. 25.2 Stereochemistry of Amino Acids Configuration of -Amino Acids Glycine is achiral. All of the other amino acids in proteins have the L-configuration at their carbon. – CO2 + H3N H R 25.3 Acid-Base Behavior of Amino Acids Recall While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively. How do we know this? Properties of Glycine The properties of glycine: high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent more consistent with this than this •• O •• + H3NCH2C •• O •• •• •– O• •• •• H2NCH2C •• OH •• Properties of Glycine The properties of glycine: high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent more consistent with this •• O •• + H3NCH2C •• •– O• •• called a zwitterion or dipolar ion Acid-Base Properties of Glycine The zwitterionic structure of glycine also follows from considering its acid-base properties. A good way to think about this is to start with the structure of glycine in strongly acidic solution, say pH = 1. At pH = 1, glycine exists in its protonated form (a monocation). •• O •• + H3NCH2C •• OH •• Acid-Base Properties of Glycine Now ask yourself "As the pH is raised, which is the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is it the proton of the carboxyl group?" You can choose between them by estimating their respective pKas. typical ammonium ion: pKa ~9 •• O •• + H3NCH2C •• OH •• typical carboxylic acid: pKa ~5 Acid-Base Properties of Glycine The more acidic proton belongs to the CO2H group. It is the first one removed as the pH is raised. •• O •• + H3NCH2C •• OH •• typical carboxylic acid: pKa ~5 Acid-Base Properties of Glycine Therefore, the more stable neutral form of glycine is the zwitterion. •• O •• + H3NCH2C •• •– O• •• •• O •• + H3NCH2C •• OH •• typical carboxylic acid: pKa ~5 Acid-Base Properties of Glycine The measured pKa of glycine is 2.34. Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an electron-withdrawing, acid-strengthening substituent on the carbon. •• O •• + H3NCH2C •• OH •• typical carboxylic acid: pKa ~5 Acid-Base Properties of Glycine A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased further. •• O •• + H3NCH2C •• •– O• •• HO – •• O •• •• H2NCH2C •• •– O• •• The pKa for removal of this proton is 9.60. This value is about the same as that for NH4+ (9.3). Isoelectric Point (pI) •• O •• + H3NCH2C •• OH •• pKa = 2.34 •• O •• + H3NCH2C •• •– O• •• pKa = 9.60 •• O •• •• H2NCH2C •• •– O• •• The pH at which the concentration of the zwitterion is a maximum is called the isoelectric point. Its numerical value is the average of the two pKas. The pI of glycine is 5.97. Acid-Base Properties of Amino Acids One way in which amino acids differ is in respect to their acid-base properties. This is the basis for certain experimental methods for separating and identifying them. Just as important, the difference in acid-base properties among various side chains affects the properties of the proteins that contain them. Table 25.2 gives pKa and pI values for amino acids with neutral side chains. Table 25.2 Amino Acids with Neutral Side Chains H Glycine + H3N C H O C O – pKa1 = 2.34 pKa2 = 9.60 pI = 5.97 Table 25.2 Amino Acids with Neutral Side Chains H Alanine + H3N C CH3 O C O – pKa1 = 2.34 pKa2 = 9.69 pI = 6.00 Table 25.2 Amino Acids with Neutral Side Chains H Valine + H3N C O C O CH(CH3)2 – pKa1 = 2.32 pKa2 = 9.62 pI = 5.96 Table 25.2 Amino Acids with Neutral Side Chains H Leucine + H3N C O C O – CH2CH(CH3)2 pKa1 = 2.36 pKa2 = 9.60 pI = 5.98 Table 25.2 Amino Acids with Neutral Side Chains H Isoleucine + H3N C O C O – CH3CHCH2CH3 pKa1 = 2.36 pKa2 = 9.60 pI = 6.02 Table 25.2 Amino Acids with Neutral Side Chains Methionine + H3N H O C C CH3SCH2CH2 pKa1 = 2.28 – pKa2 = 9.21 O pI = 5.74 Table 25.2 Amino Acids with Neutral Side Chains H Proline + H2N C O C CH2 H2C C H2 O – pKa1 = 1.99 pKa2 = 10.60 pI = 6.30 Table 25.2 Amino Acids with Neutral Side Chains H Phenylalanine + H3N C CH2 O C O – pKa1 = 1.83 pKa2 = 9.13 pI = 5.48 Table 25.2 Amino Acids with Neutral Side Chains H Tryptophan + H3N C CH2 N H O C O – pKa1 = 2.83 pKa2 = 9.39 pI = 5.89 Table 25.2 Amino Acids with Neutral Side Chains H Asparagine + H3N C H2NCCH2 O O C O – pKa1 = 2.02 pKa2 = 8.80 pI = 5.41 Table 25.2 Amino Acids with Neutral Side Chains H Glutamine + H3N C H2NCCH2CH2 O O C O – pKa1 = 2.17 pKa2 = 9.13 pI = 5.65 Table 25.2 Amino Acids with Neutral Side Chains H Serine + H3N C O C CH2OH O – pKa1 = 2.21 pKa2 = 9.15 pI = 5.68 Table 25.2 Amino Acids with Neutral Side Chains H Threonine + H3N C O C CH3CHOH O – pKa1 = 2.09 pKa2 = 9.10 pI = 5.60 Table 25.2 Amino Acids with Neutral Side Chains H Tyrosine + H3N C CH2 OH O C O – pKa1 = 2.20 pKa2 = 9.11 pI = 5.66 Table 25.3 Amino Acids with Ionizable Side Chains H Aspartic acid + H3N – C OCCH2 O C O – pKa1 = pKa2 = pKa* = pI = 1.88 9.60 3.65 2.77 O For amino acids with acidic side chains, pI is the average of pKa1 and pKa*. Table 25.3 Amino Acids with Ionizable Side Chains H + H3N Glutamic acid – C OCCH2CH2 O O C O – pKa1 = pKa2 = pKa* = pI = 2.19 9.67 4.25 3.22 Table 25.3 Amino Acids with Ionizable Side Chains H + H3N C O C O – + CH2CH2CH2CH2NH3 pKa1 = pKa2 = pKa* = pI = 2.18 8.95 10.53 9.74 Lysine For amino acids with basic side chains, pI is the average of pKa2 and pKa*. Table 25.3 Amino Acids with Ionizable Side Chains H + H3N C O C O – CH2CH2CH2NHCNH2 + NH2 Arginine pKa1 = pKa2 = pKa* = pI = 2.17 9.04 12.48 10.76 Table 25.3 Amino Acids with Ionizable Side Chains H Histidine + H3N C CH2 N NH O C O – pKa1 = pKa2 = pKa* = pI = 1.82 6.00 9.17 7.59 25.4 Synthesis of Amino Acids From -Halo Carboxylic Acids O CH3CHCOH + 2NH3 Br H2O O – CH3CHCO + NH4Br +NH3 (65-70%) Strecker Synthesis O CH3CH NH4Cl NaCN CH3CHC N NH2 1. H2O, HCl, heat 2. HO– O – CH3CHCO +NH3 (52-60%) Using Diethyl Acetamidomalonate O O C C C CH3CH2O CH3CNH H OCH2CH3 O Can be used in the same manner as diethyl malonate (Section 20.11). Example O O CH3CH2OCCCOCH2CH3 H CH3CNH O 1. NaOCH2CH3 2. C6H5CH2Cl O O CH3CH2OCCCOCH2CH3 CH3CNH O CH2C6H5 (90%) O O Example HOCCCOH –CO2 CH2C6H5 H3 N + O HBr, H2O, heat HCCOH H3N + CH2C6H5 O O (65%) CH3CH2OCCCOCH2CH3 CH3CNH O CH2C6H5 25.5 Reactions of Amino Acids Acylation of Amino Group The amino nitrogen of an amino acid can be converted to an amide with the customary acylating agents. O O O + – + H3NCH2CO CH3COCCH3 O O CH3CNHCH2COH (89-92%) Esterification of Carboxyl Group The carboxyl group of an amino acid can be converted to an ester. The following illustrates Fischer esterification of alanine. O + – + H3NCHCO CH3CH2OH CH3 HCl O Cl – + H3NCHCOCH2CH3 CH3 (90-95%) Ninhydrin Test Amino acids are detected by the formation of a purple color on treatment with ninhydrin. O O OH + + H3NCHCO– OH R O O O– O RCH + CO2 + H2O + N O O 25.6 Some Biochemical Reactions of Amino Acids Biosynthesis of L-Glutamic Acid O HO2CCH2CH2CCO2H + NH3 enzymes and reducing coenzymes – HO2CCH2CH2CHCO2 + NH3 This reaction is the biochemical analog of reductive amination (Section 21.10). Transamination via L-Glutamic Acid O – HO2CCH2CH2CHCO2 + CH3CCO2H + NH3 L-Glutamic acid acts as a source of the amine group in the biochemical conversion of -keto acids to other amino acids. In the example to be shown, pyruvic acid is converted to L-alanine. Transamination via L-Glutamic Acid O – HO2CCH2CH2CHCO2 + CH3CCO2H + NH3 enzymes O HO2CCH2CH2CCO2H – + CH3CHCO2 + NH3 Mechanism – HO2CCH2CH2CHCO2 + + NH3 PLP The first step is imine formation between the amino group of L-glutamic acid and a coenzyme called pyridoxal phosphate (PLP). Mechanism – HO2CCH2CH2CHCO2 + + NH3 HO2CCH2CH2CHCO2– Formation of the imine is followed by proton removal at one carbon and protonation of another carbon. H HO2CCH2CH2CCO2– HO2CCH2CH2CCO2– H HO2CCH2CH2CCO2– HO2CCH2CH2CCO2– Hydrolysis of the imine function gives -ketoglutarate and pyridoxamine phosphate. HO2CCH2CH2CCO2– H2O – HO2CCH2CH2CCO2 O + The pyridoxamine can do the same sequence of steps in reverse with pyruvate to generate alanine and regenerate PLP. O CH3CCO2H + – CH3CHCO2 + NH3 O CH3CCO2H + Biosynthesis of L-Tyrosine L-Tyrosine is biosynthesized from L-phenylalanine. A key step is epoxidation of the aromatic ring to give an arene oxide intermediate. – CH2CHCO2 + NH3 Biosynthesis of L-Tyrosine – CH2CHCO2 O + NH3 O2, enzyme – CH2CHCO2 + NH3 Biosynthesis of L-Tyrosine – CH2CHCO2 O + NH3 enzyme HO – CH2CHCO2 + NH3 Biosynthesis of L-Tyrosine Conversion to L-tyrosine is one of the major metabolic pathways of L-phenylalanine. Individuals who lack the enzymes necessary to convert L-phenylalanine to L-tyrosine can suffer from PKU disease. In PKU disease, Lphenylalanine is diverted to a pathway leading to phenylpyruvic acid, which is toxic. Newborns are routinely tested for PKU disease. Treatment consists of reducing their dietary intake of phenylalanine-rich proteins. Decarboxylation Decarboxylation is a common reaction of amino acids. An example is the conversion of L-histidine to histamine. Antihistamines act by blocking the action of histamine. N – CH2CHCO2 N H + NH3 Decarboxylation N CH2CH2 NH2 N H –CO2, enzymes N – CH2CHCO2 N H + NH3 Neurotransmitters – The chemistry of the brain and central nervous system is affected by neurotransmitters. Several important neurotransmitters are biosynthesized from L-tyrosine. + H3N H CO2 H H OH L-Tyrosine Neurotransmitters – The common name of this compound is L-DOPA. It occurs naturally in the brain. It is widely prescribed to reduce the symptoms of Parkinsonism. + H3N H CO2 H H HO OH L-3,4-Dihydroxyphenylalanine Neurotransmitters Dopamine is formed by decarboxylation of L-DOPA. H H2N H H H HO OH Dopamine Neurotransmitters H H2N H H OH HO OH Norepinephrine Neurotransmitters H CH3NH H H OH HO OH Epinephrine 25.7 Peptides Peptides Peptides are compounds in which an amide bond links the amino group of one -amino acid and the carboxyl group of another. An amide bond of this type is often referred to as a peptide bond. Alanine and Glycine H + H3N C CH3 H O C – O + H3N C H O C – O Alanylglycine H + H3N C CH3 H O C N C H H O C – O Two -amino acids are joined by a peptide bond in alanylglycine. It is a dipeptide. Alanylglycine H + H3N N-terminus C CH3 H O C N C H H Ala—Gly AG O C – O C-terminus Alanylglycine and glycylalanine are constitutional isomers H + H3N C C CH3 H + H3N C H H O N C H H H O C N C H CH3 O C – O Alanylglycine Ala—Gly AG – O Glycylalanine Gly—Ala GA O C Alanylglycine H + H3N C CH3 H O C N C H H O C – O The peptide bond is characterized by a planar geometry. Higher Peptides Peptides are classified according to the number of amino acids linked together. dipeptides, tripeptides, tetrapeptides, etc. Leucine enkephalin is an example of a pentapeptide. Leucine Enkephalin Tyr—Gly—Gly—Phe—Leu YGGFL Oxytocin 3 2 4 5 Ile—Gln—Asn Tyr 1 Cys N-terminus C-terminus Cys—Pro—Leu—GlyNH2 6 S 7 8 9 S Oxytocin is a cyclic nonapeptide. Instead of having its amino acids linked in an extended chain, two cysteine residues are joined by an S—S bond. Oxytocin S—S bond An S—S bond between two cysteines is often referred to as a disulfide bridge. 25.8 Introduction to Peptide Structure Determination Primary Structure The primary structure is the amino acid sequence plus any disulfide links. Classical Strategy (Sanger) 1. Determine what amino acids are present and their molar ratios. 2. Cleave the peptide into smaller fragments, and determine the amino acid composition of these smaller fragments. 3. Identify the N-terminus and C-terminus in the parent peptide and in each fragment. 4. Organize the information so that the sequences of small fragments can be overlapped to reveal the full sequence. 25.9 Amino Acid Analysis Amino Acid Analysis Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids. The mixture is separated by ion-exchange chromatography, which depends on the differences in pI among the various amino acids. Amino acids are detected using ninhydrin. Automated method; requires only 10-5 to 10-7 g of peptide. 25.10 Partial Hydrolysis of Peptides Partial Hydrolysis of Peptides and Proteins Acid-hydrolysis of the peptide cleaves all of the peptide bonds. Cleaving some, but not all, of the peptide bonds gives smaller fragments. These smaller fragments are then separated and the amino acids present in each fragment determined. Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis. Partial Hydrolysis of Peptides and Proteins The enzymes that catalyze the hydrolysis of peptide bonds are called peptidases, proteases, or proteolytic enzymes. Trypsin Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine. O O O NHCHC NHCHC NHCHC R R' R" lysine or arginine Chymotrypsin Chymotrypsin is selective for cleaving the peptide bond to the carboxyl group of amino acids with an aromatic side chain. O O O NHCHC NHCHC NHCHC R R' R" phenylalanine, tyrosine, tryptophan Carboxypeptidase Carboxypeptidase is selective for cleaving the peptide bond to the C-terminal amino acid. O O + H3NCHC R protein C O – NHCHCO R 25.11 End Group Analysis End Group Analysis Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left. We need to know what the N-terminal and Cterminal amino acids are. The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis. Several chemical methods have been developed for identifying the N-terminus. They depend on the fact that the amino N at the terminus is more nucleophilic than any of the amide nitrogens. Sanger's Method The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4dinitrobenzene. 1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution (Chapter 12). NO2 O2N F Sanger's Method 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. NO2 O2N O O F + H2NCHC NHCHC NHCH2C O2N O NHCHC O NHCHC O NHCH2C CH(CH3)2 CH2C6H5 – NHCHCO CH3 CH(CH3)2 CH2C6H5 NO2 O O O – NHCHCO CH3 Sanger's Method Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group. NO2 O O O O + + + NHCHCOH + H3NCHCO– + H3NCH2CO– + H3NCHCO– O2N CH(CH3)2 CH3 CH2C6H5 H3O+ NO2 O2N O NHCHC O NHCHC O NHCH2C CH(CH3)2 CH2C6H5 O – NHCHCO CH3 25.12 Insulin Insulin Insulin is a polypeptide with 51 amino acids. It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids). The following describes how the amino acid sequence of the B chain was determined. The B Chain of Bovine Insulin Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA The B Chain of Bovine Insulin FVNQHLCGSHL VGAL VCGERGF YTPKA The B Chain of Bovine Insulin Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALY YLVC VCGERGF YTPKA The B Chain of Bovine Insulin Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence. The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALY YLVC VCGERGF GFFYTPK YTPKA The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALY YLVC VCGERGF GFFYTPK YTPKA FVNQHLCGSHLVGALYLVCGERGFFYTPKA Insulin The sequence of the A chain was determined using the same strategy. Establishing the disulfide links between cysteine residues completed the primary structure. Primary Structure of Bovine Insulin N terminus of A chain S S C terminus of A chain 15 5 E Q C V C S L Y Q L I F E N 20 C V YC A S S 10 N S S H L N Q V S C F G S H L V G A L Y L V 5 C 15 G 20 10 N terminus E of B chain G R F F Y K P T A C terminus 25 30 of B chain 25.13 The Edman Degradation and Automated Sequencing of Peptides Edman Degradation 1. Method for determining N-terminal amino acid. 2. Can be done sequentially one residue at a time on the same sample. Usually one can determine the first 20 or so amino acids from the N-terminus by this method. 3. 10-10 g of sample is sufficient. 4. Has been automated. Edman Degradation The key reagent in the Edman degradation is phenyl isothiocyanate. N C S Edman Degradation Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid. O C6H5N C S + + H3NCHC R NH peptide Edman Degradation S O C6H5NHCNHCHC peptide NH R O C6H5N C S + + H3NCHC R NH peptide Edman Degradation S O C6H5NHCNHCHC NH peptide R The product is a phenylthiocarbamoyl (PTC) derivative. The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the peptide. Edman Degradation S O C6H5NHCNHCHC peptide NH R HCl S C6H5NH C C N CH R O + + H3N peptide Edman Degradation The product is a thiazolone. Under the conditions of its formation, the thiazolone rearranges to a phenylthiohydantoin (PTH) derivative. S C6H5NH C C N CH R O + + H3N peptide Edman Degradation C6H5 S N C C The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation. O CH HN R S C6H5NH C C N CH R O + + H3N peptide 25.14 The Strategy of Peptide Synthesis General Considerations Making peptide bonds between amino acids is not difficult. The challenge is connecting amino acids in the correct sequence. Random peptide bond formation in a mixture of phenylalanine and glycine, for example, will give four dipeptides. Phe—Phe Gly—Gly Phe—Gly Gly—Phe General Strategy 1. Limit the number of possibilities by "protecting" the nitrogen of one amino acid and the carboxyl group of the other. N-Protected phenylalanine O X NHCHCOH CH2C6H5 C-Protected glycine O H2NCH2C Y General Strategy 2. Couple the two protected amino acids. X O O NHCHC NHCH2C Y CH2C6H5 O X NHCHCOH CH2C6H5 O H2NCH2C Y General Strategy 3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus. X O O NHCHC NHCH2C Y CH2C6H5 O + H3NCHC O – NHCH2CO CH2C6H5 Phe-Gly 25.15 Amino Group Protection Protect Amino Groups as Amides Amino groups are normally protected by converting them to amides. Benzyloxycarbonyl (C6H5CH2O—) is a common protecting group. It is abbreviated as Z. Z-protection is carried out by treating an amino acid with benzyloxycarbonyl chloride. Protect Amino Groups as Amides O O CH2OCCl + + – H3NCHCO CH2C6H5 1. NaOH, H2O 2. H+ O CH2OC O NHCHCOH CH2C6H5 (82-87%) Protect Amino Groups as Amides O CH2OC O NHCHCOH CH2C6H5 is abbreviated as: O ZNHCHCOH CH2C6H5 or Z-Phe Removing Z-Protection An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by: a) hydrogenolysis b) cleavage with HBr in acetic acid Hydrogenolysis of Z-Protecting Group O CH2OC O NHCHCNHCH2CO2CH2CH3 CH2C6H5 H2, Pd O CH3 CO2 H2NCHCNHCH2CO2CH2CH3 CH2C6H5 (100%) HBr Cleavage of Z-Protecting Group O CH2OC O NHCHCNHCH2CO2CH2CH3 CH2C6H5 HBr O CH2Br CO2 + H3NCHCNHCH2CO2CH2CH3 – CH2C6H5 Br (82%) The tert-Butoxycarbonyl Protecting Group O (CH3)3COC O NHCHCOH CH2C6H5 is abbreviated as: O BocNHCHCOH CH2C6H5 or Boc-Phe HBr Cleavage of Boc-Protecting Group O (CH3)3COC O NHCHCNHCH2CO2CH2CH3 CH2C6H5 HBr O H3C C H3C CH2 CO2 + H3NCHCNHCH2CO2CH2CH3 – CH2C6H5 Br (86%) 25.16 Carboxyl Group Protection Protect Carboxyl Groups as Esters Carboxyl groups are normally protected as esters. Deprotection of methyl and ethyl esters is by hydrolysis in base. Benzyl esters can be cleaved by hydrogenolysis. Hydrogenolysis of Benzyl Esters O O C6H5CH2OC O NHCHCNHCH2COCH2C6H5 CH2C6H5 H2, Pd O C6H5CH3 CO2 + – H3NCHCNHCH2CO CH2C6H5 (87%) CH3C6H5 25.17 Peptide Bond Formation Forming Peptide Bonds The two major methods are: 1. coupling of suitably protected amino acids using N,N'-dicyclohexylcarbodiimide (DCCI) 2. via an active ester of the N-terminal amino acid. DCCI-Promoted Coupling O O ZNHCHCOH + H2NCH2COCH2CH3 CH2C6H5 DCCI, chloroform O ZNHCHC O NHCH2COCH2CH3 CH2C6H5 (83%) Mechanism of DCCI-Promoted Coupling O + ZNHCHCOH C6H11N C CH2C6H5 H C6H11N O C C6H11N OCCHNHZ CH2C6H5 NC6H11 Mechanism of DCCI-Promoted Coupling The species formed by addition of the Zprotected amino acid to DCCI is similar in structure to an acid anhydride and acts as an acylating agent. Attack by the amine function of the carboxylprotected amino acid on the carbonyl group leads to nucleophilic acyl substitution. H C6H11N O C C6H11N OCCHNHZ CH2C6H5 Mechanism of DCCI-Promoted Coupling O H C6H11N C O + ZNHCHC O NHCH2COCH2CH3 CH2C6H5 C6H11NH O H2NCH2COCH2CH3 H C6H11N O C C6H11N OCCHNHZ CH2C6H5 The Active Ester Method A p-nitrophenyl ester is an example of an "active ester." p-Nitrophenyl is a better leaving group than methyl or ethyl, and p-nitrophenyl esters are more reactive in nucleophilic acyl substitution. The Active Ester Method O O ZNHCHCO NO2 + H2NCH2COCH2CH3 CH2C6H5 chloroform O ZNHCHC O NHCH2COCH2CH3 + HO CH2C6H5 (78%) NO2 25.18 Solid-Phase Peptide Synthesis: The Merrifield Method Solid-Phase Peptide Synthesis In solid-phase synthesis, the starting material is bonded to an inert solid support. Reactants are added in solution. Reaction occurs at the interface between the solid and the solution. Because the starting material is bonded to the solid, any product from the starting material remains bonded as well. Purification involves simply washing the byproducts from the solid support. The Solid Support CH2 CH CH2 CH CH2 CH CH2 CH The solid support is a copolymer of styrene and divinylbenzene. It is represented above as if it were polystyrene. Cross-linking with divinylbenzene simply provides a more rigid polymer. The Solid Support CH2 CH CH2 CH CH2 CH CH2 CH Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings. The Solid Support CH2 CH CH2 CH CH2 CH CH2 CH CH2Cl The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2). The Solid Support CH2 CH CH2 CH CH2 CH CH2 CH CH2Cl The chloromethylated resin is treated with the Bocprotected C-terminal amino acid. Nucleophilic substitution occurs, and the Boc-protected amino acid is bound to the resin as an ester. The Merrifield Procedure CH2 CH CH2 CH CH2 O – BocNHCHCO R CH CH2 CH2Cl CH The Merrifield Procedure CH2 CH CH2 CH CH2 O BocNHCHCO Next, the Boc protecting group is removed with HCl. R CH CH2 CH2 CH The Merrifield Procedure CH2 CH CH2 CH CH2 O H2NCHCO DCCI-promoted coupling adds the second amino acid. R CH CH2 CH2 CH The Merrifield Procedure CH2 CH CH2 CH CH2 O BocNHCHC R' CH O CH2 CH CH2 NHCHCO R Remove the Boc protecting group. The Merrifield Procedure CH2 CH CH2 CH CH2 O H2NCHC R' CH O CH2 CH CH2 NHCHCO R Add the next amino acid and repeat. The Merrifield Procedure CH2 CH CH2 O CH CH2 O + H3N peptide C NHCHC R' CH O CH2 CH CH2 NHCHCO R Remove the peptide from the resin with HBr in CF3CO2H. The Merrifield Procedure CH2 CH CH2 CH CH2 CH CH2 CH2Br O O + H3N peptide C NHCHC R' O – NHCHCO R CH The Merrifield Method Merrifield automated his solid-phase method. Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in 68% yield. Synthesized ribonuclease (124 amino acids) in 1969. 369 reactions; 11,391 steps Nobel Prize in chemistry: 1984 25.19 Secondary Structures of Peptides and Proteins Levels of Protein Structure Primary structure = the amino acid sequence plus disulfide links. Secondary structure = conformational relationship between nearest neighbor amino acids. helix pleated sheet Levels of Protein Structure The -helix and pleated sheet are both characterized by: Planar geometry of peptide bond Anti conformation of main chain Hydrogen bonds between N—H and O=C Pleated Sheet Shown is a sheet of protein chains composed of alternating glycine and alanine residues. Adjacent chains are antiparallel. Hydrogen bonds between chains. van der Waals forces produce pleated effect. Pleated Sheet Sheet is most commonly seen with amino acids having small side chains (glycine, alanine, serine). 80% of fibroin (main protein in silk) is repeating sequence of —Gly—Ser—Gly—Ala—Gly—Ala—. Sheet is flexible, but resists stretching. Helix Shown is an helix of a protein in which all of the amino acids are L-alanine. Helix is right-handed with 3.6 amino acids per turn. Hydrogen bonds are within a single chain. Protein of muscle (myosin) and wool (-keratin) contain large regions of -helix. Chain can be stretched. 25.20 Tertiary Structure of Polypeptides and Proteins Tertiary Structure Refers to overall shape (how the chain is folded). Fibrous proteins (hair, tendons, wool) have elongated shapes. Globular proteins are approximately spherical. Most enzymes are globular proteins. An example is carboxypeptidase. Carboxypeptidase Carboxypeptidase is an enzyme that catalyzes the hydrolysis of proteins at their C-terminus. It is a metalloenzyme containing Zn2+ at its active site. An amino acid with a positively charged side chain (Arg-145) is near the active site. Carboxypeptidase Disulfide bond Zn2+ Arg-145 N-terminus C-terminus Tube model Ribbon model What Happens at the Active Site? •• • O• + H3N peptide C O NHCHC – R O H2N + C H2N Arg-145 What Happens at the Active Site? •• • O• + H3N peptide C O NHCHC – R O H2N + C Arg-145 H2N The peptide or protein is bound at the active site by electrostatic attraction between its negatively charged carboxylate ion and arginine-145. What Happens at the Active Site? •• • O• + H3N peptide C Zn2+ O NHCHC – R O H2N + C Arg-145 H2N Zn2+ acts as a Lewis acid toward the carbonyl oxygen, increasing the positive character of the carbonyl carbon. What Happens at the Active Site? Zn2+ •• • O• + H3N peptide C O NHCHC – R O H2N + C Arg-145 H2N H • O• • • H Water attacks the carbonyl carbon. Nucleophilic acyl substitution occurs. What Happens at the Active Site? Zn2+ H2N + C •• • O• + H3N peptide C •• – O •• •• H2N O + H3NCHC – R O Arg-145 25.21 Coenzymes Coenzymes The range of chemical reactions that amino acid side chains can participate in is relatively limited. Acid-base (transfer and accept protons) Nucleophilic acyl substitution Many other biological processes, such as oxidation-reduction, require coenzymes, cofactors, or prosthetic groups in order to occur. Coenzymes NADH, coenzyme A and coenzyme B12 are examples of coenzymes. Heme is another example. Heme H 2C CH H 3C CH3 N N CH CH2 Fe N H 3C HO2CCH2CH2 N CH3 CH2CH2CO2H Molecule surrounding iron is a type of porphyrin. Myoglobin C-terminus Heme N-terminus Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen transport). 25.22 Protein Quaternary Structure: Hemoglobin Protein Quaternary Structure Some proteins are assemblies of two or more chains. The way in which these chains are organized is called the quaternary structure. Hemoglobin, for example, consists of 4 subunits. There are 2 chains (identical) and 2 chains (also identical). Each subunit contains one heme and each protein is about the size of myoglobin. 25.23 G-Coupled Protein Receptors G-Coupled Protein Receptors GCPRs (the “G” stands for guanine in “guanine nucleotide-binding proteins”) occur throughout the body and function as “molecular switches” that regulate many physiological processes. GCPRs span the cell membrane and when they bind their specific ligand (a small organic molecule, lipid, peptide, ion, etc.), they undergo a conformational change, which results in the transduction of a signal across the membrane. The Core of Modern Medicine GCPRs are the target for many therapeutic agents in the treatment of cancer, cardiac malfunction, inflammation, pain, obesity, diabetes and disorders of the central nervous system.