hapter 3 Amino Acids αamino Acids are the main constituents of proteins. But they also can function as neurotransmitters (glutamate, γaminobutyric acid), hormones (thyroxine; see right), as bacterial cell wall components (Dalanine) and are intermediates in many metabolic pathways e.g. Glycine is a precursor to heme. ormones are chemical messengers secreted into the bloodstream by one cell that affect the metabolism of another cell. I I 2 I I They may be hydrophilic such as amino acids and peptides or they may be hydrophobic such as steroids. Side hain Formula for αaa at p 7 R α 2 base high p form α acid low p form αarbon A Protein is a Biopolymer composed of a linear chain of αaa. nly 20 αamino acids are encoded in genetic material DA. A Peptide is a short protein, up to 20 AA. 1
AA Sidehain lassification: A. Aliphatic, nonpolar, hydrophobic 3 3 Leucine Leu 2 3 Isoleucine Ile 3 2 Valine Val 3 3 Proline Pro A cyclic αimino acid. 2 2 2 2 2
Alanine Ala 3 Glycine Gly B. Aromatic Amino Acids in order of hydrophobicity They absorb ultraviolet light because of conjugated double bonds. 3
Phenylalanine Phe 2 Tryptophan Trp Indole 2 Tyrosine Tyr Phenolic hydroxyl pk a = 10.1 2. Sulphurcontaining AA 3 S Methionine Met 2 2 4
ysteine ys S 2 Thiol pk a ~ 8.3 Two nearby ys can form a disulphide bond in an oxidizing environment. S S 2 2 D. ydroxyl Amino Acids polar Threonine Thr Alcohol pk a = 13.6 3 Serine Ser Alcohol pk a = 13.6 2 5
E. Amidecontaining sidechains 2 Glutamine Gln 2 2 Amide sidechains do not ionize 2 Asparagine Asn 2 F. Acidic AA (negative charge in the sidechain at p 7) Glutamic Acid Glu arboxyl pk a = 4.2 2 2 Aspartic Acid Asp arboxyl pk a = 3.9 2 6
D. Basic (Positive charge in the sidechain ) 3 2 Lysine Lys 2 2 εamino pk a = 10.5 2 2 2 Arginine Arg 2 Guanidino pk a = 12.5 2 2 istidine is Imidazole pk a = 6.0 2 istamine 2 2 3 Side chains in Groups A and B carry no net electric charge at p 7 and remain stationary in an electric field. 7
R Zwitterion hybrid ion. 3 Average values for AA carboxyl and amino pk a s are 2.2 and 9.5. At p 7, acidic amino acids have lost a proton from their side chains and are anionic. They move toward a positive electrode. At p 7, basic amino acids bind a and are cationic. They move toward a negative electrode. 2 3 Molecular onformation refers to differences in the spatial arrangement of groups joined by covalent bonds due to bond rotation. Molecular onfiguration refers to isomers that can be interconverted only by breaking bonds. Isomers have the same molecular formula but a different arrangement of the groups. e.g. Leu, Ile. All AA except Glycine, have 4 different groups attached to the tetrahedral α. The α is asymmetric / chiral (handed). 8
There are 2 possible arrangements of the 4 groups called stereoisomers. They are nonsuperimposable mirror images / enantiomers. 3 3 LAla 3 3 DAla D and L define the absolute configuration using D and Lglyceraldehyde. (RS is also used.) D and Lglyceraldehyde are reference molecules for assignment of stereochemistry (absolute configuration). 2 LGlyceraldehyde 2 DGlyceraldehyde The aldehyde and acid are aligned and the hydroxyl and amino groups are aligned. These molecules are optically active: they rotate the plane of monochromatic planepolarized light in opposite directions. dextrorotatory () or levorotatory (). 9
All AA in proteins are L. Some D are found in antibiotic peptides. Thr and Ile contain two chiral s and 4 stereoisomers. 3 3 3 2 ere are 2 stereoisomers that are not enantiomers. Maleic Acid cis Fumaric Acid trans Thalidomide is a sedative. The form is therapeutic, the mirror image is teratogenic and causes embryo malformation. 3 2 3 2 3 ()limonene fresh citrus orangelike Titration of Gly with : The titration curve will have two buffering regions, one for each group. 3 ()limonene harsh, turpentine like, lemon Amino pk a = 9.6 arboxyl pk a = 2.3 Average values for AA carboxyl and amino pk a s are 2.2 and 9.5. 10
Draw Gly at low p (). Add and raise the p to 2.3: The strongest acid (lowest pk a ) will ionize first. 3 2 After adding 0.5 equivalents of, p = pk a1 = 2.3 and [] = [ ] and [] = [Z]. 3 2 3 2 After adding 1.0 equivalent of, [ ] = [Ala], the carboxyl is Z 100% ionized, Gly is Zwitterionic, and the p is midway between 2.3 and 9.6. p = = 6.0 = pi 2.3 9.6 2 The Isoelectric Point is the p at which the concentration of the Zform of an AA is maximum. n average there is no net charge on the AA. At the pi, the AA is stationary in an electric field. 11
After addition of 1.5 equivalents of, p = pk a2 = 9.6 and [ 3 ] = [ 2 ] and A = Z. After addition of 2.0 equivalents of, the p ~ 12 and Gly is 100% A. 3 2 3 2 Z 2 2 A otes: 1. Region between buffering regions is not a good buffering region. 2 2 2. eutral AA never exist. 12 other AA with nonionizable side chains have titration curves just like Gly and Ala. Titration of Glutamic Acid p There are 3 buffering regions centred on p 2.2, 4.25, and 9.7. 14 12 10 8 6 4 2 2.2 4.25 9.7 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 mole equivalents of 2 2 12
What is the pi? At p 2.2 there is 50%, 50% Z. At p 4.3 there is 50% Z, 50% A1. At p 9.7 there is 50% A1, 50% A2 Glu 3 Glu 3 2.2 A1 Z 4.3 Glu 3 So 1/2 way between 4.3 and 2.2 there will be 100% Z. So pi = 2.2 4.3 2 = 3.25 A2 9.7 2 Glu Titration of is: There are 3 buffering regions centred on p 1.8, 6.0, and 9.2. 13
At p 1.8 [1] = [2] At p 6.0 [2] = [Z] At p 9.1 [Z] = [A] What is the pi? pi = 9.1 6.0 2 3 = 7.5 2 1 1.8 Z 6.0 2 2 3 Sample Buffer Question: To 50 ml of 0.1 M acetate buffer p 4.75, is added 1 ml of 0.2 M l. What is the new p? The pk a of acetic acid is 4.75. A 9.1 2 2 2 3 Sketch and label the titration curve: What do I know? p = 4.75 = pk a, so [A ] = [A] [A ] [A] = 0.1 M In 50 ml there are 5 millimoles of buffer; 2.5 millimoles = A = A. 1 ml of 0.2 M l contains 0.2 millimoles of. Deduce the above yourself A A p = pk a log{ [A ] [A] } 14
0.2 millimoles of l will neutralize 0.2 millimoles of buffer (A ). i.e. 0.2 react with 0.2 A to form 0.2 A. So A new = 2.5 0.2 = 2.7 millimoles. A new = 2.5 0.2 = 2.3 millimoles. Plug into : [2.3mmoles / 51ml] p = 4.75 log{ [2.7mmoles / 51ml] } = 4.75 log{0.85} = 4.75 0.07 = 4.68 ear the pk a, a small addition of has caused only a small change in p A second question: To 50 ml of 0.1 M acetate buffer, p 2.75, is added 1 ml of 0.2M a. What is the new p? ere we cannot assume that we have equal amounts of A and A. Plugging into : Take the anitlog of both sides gives: ow [A] 10 2 = [A ] 2.75 = 4.75 log{ [A ] and [A] } 2.0 = log{ [A ] [A] } We also know that, A A = 5.0 millimoles So 0.01 A A = 5.0 millimoles 10 2 = [A ] [A] 15
A = 4.95 millimoles so A = 5.0 4.95 = 0.05 millimoles. This is what we started with. ow, what happens when is added? 0.2 millimoles of will neutralize 0.2 millimoles of A. i.e. 0.2 0.2 A à 0.2 A 0.2 2 So A new = 4.95 0.2 = 4.75 millimoles and A new = 0.05 0.2 = 0.25 millimoles. Plug into : [0.25mmoles / 51ml] p = 4.75 log{ [4.75mmoles / 51ml] } = 4.75 log {0.053} = 4.75 1.28 = 3.47 Far from the pk a, a small addition of has caused a large change in p. Amino acids, proteins, and nucleic acids are often purified using methods that separate the molecules based on differences in net charge. Electrophoresis separates molecules by applying an electric field. Ions move in the field according to their net charge. In paper electrophoresis the ions move through a buffer solution that permeates the paper. e.g. a mixture of Ala, Lys, and Asp at p 6 will have different charges. 16
AA et harge Ala 0 Lys Asp Ala will be stationary, Lys will move toward the cathode () and Asp will move toward the anode (). Filter paper soaked in buffer at p 6.02 Spot a mixture of AA AA begin to separate. 17
After electrophoresis Lys Ala Asp Amino acids can also be separated in thin silica capillaries apillary Electrophoresis. IonExchange hromatography Polystyrene or silicabased beads have anionic or cationic functional groups attached. S 3 3 3 ation Exchanger Anion Exchanger Sulfonic acid is a strong acid and is anionic at p 2 whereas all amino acids are cationic. Beads with sulphonic acid are packed into a cylindrical column. A mixture of AA is applied in a buffer at p 2 and bind to the sulphonic acid. 18
The AA are removed by washing the beads with buffers at higher p. When the p reaches the pi of an AA it unbinds from the beads and washes out of the column. Differences in the pi of each AA cause them to be dissociated at different ps. ere is a typical elution profile: p 2 p 2 p 2.77 All AA () bound Asp ( ) unbinds Peptides The condensation of AA does not occur in neutral water as the is not a good leaving group. 3 R R' condensation You will learn how cells do this in EM/MBI 2780. R R' 2 3 Polypeptide: more than 20 AA. Protein: 1 or more polypeptides. Amide / Peptide bond AlanylSerylLysylGlutamate is a tetrapeptide. 19
At p 7 its structure is: 3 Backbone 3 otes: Amino Terminus 2 ( 2 ) 4 ( 2 ) 2 Internal "Residues" Sidechains 3 arboxyl Terminus 1. Repeating backbone is nonionizable, except the ends. 2. Sidechains vary. 3. pk a, pi of side chains and termini are similar to the AA. 4. Amino terminus at left, carboxyl terminus at right. BiologicallyActive Peptides Enkephalins are natural analgesics. They bind to specific receptors in the brain and diminish the perception of pain. e.g. TyrGlyGlyPheMet Peptides can adopt many conformations because of rotation about single covalent bonds. Binding to a target receptor may force them to adopt a single conformation. 20
Aspartame: a synthetic dipeptide 150 X sweeter than sugar. 2 3 2 3 Methyl ester 21