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1、Chapter 3 Amino acids and PeptidesBiochemistry lecture 2 (Sept. 13, 2012)a-amino acid20 standard a a-amino acids serve as building blocks of all proteins All have both an amino and a carboxyl group linked to their a a-carbons (chiral). The amino acids are categorized according to features of their s
2、ide chains, which vary in size, shape, polarity, charge, H-bonding capacity, hydrophobicity, chemical reactivity, etc.a a1. Nonpolar and aliphatic;2. Aromatic; 3. Polar and uncharged;4. Positively charged;5. Negatively charged. 1.Aliphatic;2.Hydroxyl or sulfur-containing3. Cyclic;4. Aromatic;5. Basi
3、c6. Acidic and amide1. Electrically charged; 2. polar uncharged;3. Special cases; 4. hydrophobic.Each has: a common name; a three-letter abbreviation;a one-letter symbol.Some are designated phoneticallyYou must memorize all of them!Three-LetterOne-LetterAmino AcidSymbolSymbolAlanineAlaAArginineArgRA
4、sparagineAsnNAspartateAspDCysteineCysCGlutamateGluEGlutamineGlnQGlycineGlyGHistidineHisHIsoleucineIleILeucineLeuLLysineLysKMethionineMetMPhenylalaninePheFProlineProPSerineSerSThreonineThrTTryptophanTrpWTyrosineTyrYValineValVSelenocysteine and pyrrolysine were recently found to be proteinogenic amino
5、 acidsGlutathione peroxidase isa selenoprotein(in all kingdoms of life) Selenocysteine tRNA is made from Ser-tRNASec Monomethylamine methyltransferase contains Pyl(an enzyme involved inmethane production inmethanogenic archaea) Uses a novel tRNA:aminoacyl-tRNAsynthetase pair Selenocysteine (Sec, U)(
6、Encoded by the UGA codon)Pyrrolysine (Pyl, O)(encoded by the UAG codon)Discovered in 2002Discovered in 1976Hao et al. (2002) Science 296,14621466.Cone et al. (1976) PNAS 73: 26592663 The 20 standard proteogenic amino acids were revealed over a long period of time Proteins are decomposed into relativ
7、ely simple substances upon hydrolytic action of boiling acids (1830s). Such products are the mixture of components of similar properties (extremely difficult to separate!). Enzyme digestion of proteins (much milder condition) helped to unveil which of the amino acids are building blocks of proteins.
8、 It took about 100 years (1830s-1930s) for the complete identification of all the amino acid building blocks of proteins (Partition chromatography greatly helped to resolve this issue). Early methods to isolate the amino acids from degraded proteins Chance products of fractional crystallization (Leu
9、, Gly, Ala, Tyr, Phe, Glu, and Ser). Precipitation of different derivatives of particular amino acids (Asp, Lys, Arg, His). Following the color reaction (Trp). Requirements for bacterial growth (Met). Other unusual methods (e.g., Cystine from urine calculus. etc.Homochirality of the standard amino a
10、cids: Only L-amino acids are found in proteins D-, L- Convention(Emil Fischer, 1891)The correlation of structure (or configuration) with optical rotation is very complex and has not been successful to date! (i.e., the D-and L-signs do not tell anything about their optical rotation!)All rotating plan
11、e-polarized light (1900s, Emil Fischer);Similarity to glyceraldehyde is used to designate configurationA pairs of enantiomersThe aromatic amino acids allow peptides (proteins) to absorb light in the near UV region (280 nm) Trp and Tyr contain delocalized pai electrons and strongly absorb UV light (a
12、round 280 nm). Protein (peptide) concentration can be measured at 280 nm using the Lambert-Beer Law: A =log (I0/I)= e eclA- absorbance of light at a given wavelength;e-e-extinction coefficient;C- molar concentration;l- length of light passD-configuration of these amino acids are also found in nature
13、 In the peptidoglycan of bacterial cell walls Certain short peptides (e.g., conotoxin, antibiotics).H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2 -conotoxin Free amino acids are predominantly in zwitterion form at cellular neutral pH range
14、Net charge: +1 0 -1(pH between 2.2 and 9.4) Amino acids exist as zwitterions in solid phase, crystallize with salt-like properties unlike typical organic acids or amines (thus having high melting points)Free amino acids behave as di-or tri-protonic acids Acid dissociation constant: HA A + H+,The Ka
15、values would span many orders of magnitude!The larger the value of pKa, the smaller the extent of dissociation, the weaker the acid. The neighboring carboxyl and amino groups affect each other for their dissociationEach amino acid (or peptide, protein) has a characteristic isoelectric point (pI) val
16、ue pI: the pH at which a molecule carries no net electric charge (would not migrate in an electric field). For non-charged amino acids: pI = (pK1 + pK2) Side chain: uncharged.Each amino acid (or peptide, protein) has a characteristic isoelectric point (pI) value For acidic amino acids: pI = (pK1 + p
17、KR) Side chain: negatively-chargedEach amino acid (or peptide, protein) has a characteristic isoelectric point (pI) value For basic amino acids: pI = (pKR + pK2)Net chargeSide chain: positively-chargedAmino AcidShortAbbrev.Avg. Mass (Da)pIAlanineAAla89.06.0CysteineCCys121.15.1Aspartic acidDAsp133.12
18、.9Glutamic acidEGlu147.13.2PhenylalanineFPhe165.15.5GlycineGGly75.066.1HistidineHHis155.27.6IsoleucineIIle131.26.1LysineKLys146.29.6LeucineLLeu131.16.0MethionineMMet149.25.7AsparagineNAsn132.15.4ProlinePPro115.16.3GlutamineQGln146.15.7ArginineRArg174.210.8SerineSSer105.15.7ThreonineTThr119.15.6Valin
19、eVVal117.16.0TryptophanWTrp204.25.9TyrosineYTyr181.15.6Amino acids having very different pI values would have different charges at a particular pH, and can be separated from each other by ion-exchange chromatography.Some amino acids can be modified after being incorporated into specific proteins, wh
20、ich further increases the diversity of protein structure and propertiesCH 3 SCH 2 CH 2 CHNHCOHCOA few examples: (sugar and lipidscan also be covalentlyadded to specific amino acid residues in proteins)There are about 200 such modifications in human proteinsRepresentative modificationsof the standard
21、 amino acids:No modifications on the non-polar(hydrophobic amino acids)The chromophore group in GFP is formed from amino acid residues!Hundreds of non-proteinogenic amino acids found in living organisms They have other biological functions (many yet revealed)Non-natural amino acids can be introduced
22、 into proteins as probes to unveil protein behavior in living cellsMeng Zhang et al., 2011, Nat. Chem. Biol. 7:671-677.Two of thesenon-natural amino acidsSuch introduced non-natural amino acids will allow photo-crosslinking to occur in living cellsNeeded:A transfer-RNA: aminoacyl tRNA-synthetase pai
23、r;A newly-created unique codon;The non-natural amino acid. Peptone was once considered as a form of protein present during animal digestion Recognized as products of proteins upon action of gastric juice (pepsin) and pancreatic secretion (trpsin), mainly by physiologists. Noncoagulable by heat and d
24、iffusible through membranes. Considered to be end products in gastrointestinal digestion of proteins and precursors of blood proteins (the “peptone doctrine”). Chemists failed to separate them into definable substances. Extensive digestion (with other enzymes in the digestive system) resulted in the
25、 eventual production of amino acids, and they can replace intact protein as animal nutrient.Nature of chemical linkage between amino acids in proteins unveiled via two approaches The Fischer-Hofmeister theory of protein structure: amino acids linked via amide bonds (1902).Emil FischerFranz Hofmeiste
26、rvia peptide synthesisOrganic chemistPhysiologistvia protein degradationReached this conclusion via deduction!Nobel Prize 1902(work on sugar and purines)Biuret (NH2-CO-NH-CO-NH2) reaction of peptides Effects of salts on protein solubilityAmino acids join together to form linear peptides (via condens
27、ation reaction)Dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, octapeptide, nonapeptide, decapeptide, etc.; Size boundaries from oligopeptide to polypeptide to protein is arbitrary!Ways to name peptides:SerylglysyltyrosylleucineSer-Gly-Tyr-Ala-Leu or SGYALAverage MW of an amino acid
28、residue : 128-18 = 110Peptide means “digested”.The Amino acids can be linked in a great number of different sequences(e.g., a tripeptide has 3 x 2 x 1 = 6 arrangements)Ala Ser AspAla Asp SerSer Ala AspSer Asp AlaAsp Ser AlaAsp Ala SerFor a peptide made of 20 amino acids:There is a 2.43 x 1018 possib
29、le sequencesMany short peptides have important usage or biological activities Ribosomal peptides: Hormones (e.g., vasopressin, oxytocin, gastrin), antibiotics (e.g., Microcins) Nonribosomal peptides: Glutathione (antioxidant) Short peptides can be used for antibody generation, for isolating receptor
30、s of peptide signal molecules, serve as drugs, protein structure studies.VasopressinOxytocinGastrin-14GlutathioneMicrocinAmino acid sequence can be determined by Edman degradation (1950) The residues, as phenylthiohydantoin (PTH)-amino acid derivatives, are sequentially removed one at a time from th
31、e N-Term; sequence up to 30 residues.Standard elutionPositions of the 20 PTH-amino acidPehr Edman(1916-1977) A automated protein sequenatorBasic (pH 8)Mildly acidicIdentified using chromatography or electrophoresis. Uncharged Peptides can be effectively synthesized via solid-phase peptide synthesis
32、(Merrifield, 1963) J. Amer. Chem. Soc. (1963) 85: 2149-2154. R. Bruce Merrifield (1921-2006)Nobel Prize in 1984 Unintended reactions have to be prevented With protecting groups; Proceeds in a C-terminal to N-terminal fashion (the opposite of protein biosynthesis). The peptide is immobilized on the s
33、olid-phase, liquid-phase reagents and by-products can be effectively washed away at each cycle of synthesis. Random peptide libraries could be generatedAnd used for selecting bioactive peptides.Solid resinWhy are proteins made of the 20 L-amino acids? No compelling chemical or physical reasons. The
34、synthesized D-amino acid protein was found to be physically identical. The 20 chosen ones are not the most naturally abundant or the most easily synthesized or the most chemically versatile! The first amino acids might be racemic mixtures of both D- and L-configuration. This puzzle awaits more conte
35、mplate by biochemists. Future studies of amino acids and peptides? Isolation of new bioactive amino acids and peptides. Understand the function of them. Modify and design new ones. etc.Quiz 1 The quantitative differences in biological activity between the two enantiomers of a compound are sometimes quite large. For example, the D isomer of the drug isoproterenol, used to treat mild asthma, is 50 to 80 times more effective as a bronchodilator（支氣管擴(kuò)張劑）than the L isomer. Why do the two enantiomers have such radically different bioactivity?
生物化學(xué)：Chapter 3 Amino acids and Peptides
