‘ving Acids and Proteine 2 Edman Degradation iti - _ a She K Ht Ky os oN eee EeaTIDE s ne ~ PEPTIDE | s ae ~) PEPTIDE Ba Og A wt hk tL ah t OW velop adion protontransfer | Nphenythiowea ‘ay or0up fom the N-terminal aio acid othe elactrophise carbon of phen isothiocyanate fluted by * onylthiouea. » bond from the N-terminal amino acid pentce chan that contains one fewer amino aid than the original peptide nan N-phonyihiohydantin PTH © © Co, rei A nh — several steps Cotte, Hen? ~W rar trazoinene phenytiohyanton ‘ers 12 reaction the thazolinone rearranges by @ multistep pathway to form an N-phenyIthiohydartoin (PTH) »ofiginal N-terminal amino aci. In theory a protein of any Jength can be sequenced using the Edman degradation, but in practice, the accumulation of small quantities of unwanted by-produets limits sequencing to proteins hav ing fewer than approximately 50 amino acids. 8.19 Draw the structure ofthe N-phenythichydantoin formed by inal Edman degradation of ach opti: (a) Ala-Gly-Pho-Phe; 2) Vallle-Tyc 6C Partial Hydrolysis of a Peptide ‘Additional structural information can be obtained by cleaving some, but not all, of the amide bbonds in a peptide. Partial hydrolysis of a peptide with acid forms smaller fragments in arandom fashion. Sequencing these peptides and identifying sites of overlap can be used to determine the sequence of the complete peptide, as shown in Sample Problem 28.2,Sample Problem 28.2 Problem 28.20 Problem 28.21 28.8 Peptide Sequencing 1093 ive the amino acid sequence of @ hexapeptde that contains the amino acids Ala, Vl, Serle, Gly, “Tt, ana forms the flowing fragments winen partally hydro}yzed with HC Gly-lo-Val, Ala-Ser-Gly, and Tyr-Ala Solution Looking for points of overap in the saquences ofthe small fragments shows how the fragments ‘should be pieced together In this exemple, the fragment Ala-Ser-Giy contains amino acids ‘common to the two other fragments, thus showing how the thre fragmenta can be joined together. ‘common amino acide tyfaia] — [eytn Anawor: Aled Seraly Tyr Ale-Ser-Gly-tle—Val hexapepte Give the amino acid sequence of an octapeptide that contains the amino acids Ty, Ala, Leu (2 equiv), Oys, Gly, Glu, and Val, and forms the folowing fragments when partaly hydrolyzed with HCI: Val-Cye-Giy-Glu, Ala-Lou-Tyr, and Tyr-Leu-Val-Cys. Peptides can also be hydrolyzed at specifi sites using enzymes. The enzyme carboxypeptidase catalyzes the hydrolysis of the amide bond nearest the C-terminal end, forming the C-terminal ‘amino acid and a peptide with one fewer amino acid. In this way, carboxypeptidase is used to identify the C-terminal amino acid, Other enzymes catalyze the hydrolysis of amide bonds formed with specific amino acids. For example, trypsin catalyzes the hydrolysis of amides with carbonyl group thats part ofthe basic * amino acids arginine and lysine. Chymotrypsin hydrolyzes amides with carbonyl groups that are Pat of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Table 28.2 summaizes these enzyme specificities used in peptide sequencing Chymotrypsin cleaves her Cartonypoptidace cleaves here. Aa-Pre?Ghy-Leu-fp al-ArgpHs-Pro-ProtGiy Trypsin cleaves here. Table 28.2 Cleavage Sites of Specific Enzymes in Peptide Sequencing Carvonpeptidase “Arid Bond nearest tothe C-terminal amino acid Crymotypsn “Amie bord witha carbonyl group trom Pe, Tyr, oT Topin Ande bond wih carbon ooup fom Ago lye (a) What products are formed wien each peptide Is treated with typeln? b) What products are formed when each peptida is treated with chymotypein? [1] @y-Ala-Phe-Lou-Lys-Ala [2] Phe-Tyr-Gly-Cys-frg-Sar [9] The-Pro-Lys-Glu-His-Gly-Phe-Cys-Trp-Val-Val-Pho1094 Chapter 28 Amino Acids and Proteins Sample Problem 28.3 Deducs the sequence ofa pentapeptide that contains the amino acids Ala, Gl, Gly, Soy and Ty, ‘rom the following experimental data. Edman degradation cleaves Gly from the pentapeptide, and carboxypeptidase forms Ala and a tetrapeptide, Treatment ofthe pentapeptide with chymotypsin forms a cipeptie and a tripeptide, Petal hyaroysis forms Giy, Set and tne tripeptide Tyr-GIu-Ala. Solution Use each result to determine the locaton of an amino acd in the pentapeptide. Experiment osutt + Edman degradation identifies the Nterminal amino acid—in this, > Giy-_-_-_-_ ‘case, Gy + Carbexypepticase identities the C-terminal amino acid (Ala) 3 Gy-_-_-_-Aa wen its cleaved from the end of the chain. + Chymotiypsin cleaves amide bonds that contain a carbonyl group > GiyTyr=_=_~Ala from an aromatic ano acid~Tyr i this case. Because a dipeptide o ‘and tipeptie are obtained after treatment with chymotrypsin, Ty Giy-_ “Tyre Ala ‘must be the C-terminal amino acd of ether the d-o tripeptide. {AS aresul, Tyr must be either the socond or third amino acid inthe pentapeptide chain. + Partial hydrolyls forms the tripeptide Tyr-Giu-Ala. Because Alas the C-terminal amino aci, this result identiis the last three amino .2¢ids in the chain. +The last amino acid, Sex, must be located atthe only remaining > Gly-SerTyr- Gila postion, the second amino acd in the pentapeptide, andthe ‘complete sequence is determined Problem 28.22 Deduce the sequence ofa heptapeptide tnat contains the amino acids Ale, Avg, Glu, Gy, Leu, Phe, and Ser, from the following experimental data. Edman degradation cleaves Leu trom the hheplaceptice, and carboxypeptidase forms Giu and a hexapeptde. Treatment ofthe neptapeptide with chymotrysin forms a hexapeptide and a single amino acid Treatment of the heptapeptide with trypsin forms a pentapeptide and a dipeptide. Partial hydrolysis forms Glu, Leu, Phe, and the tripoptides Gy-Ala-Ser and Ala-Ser-Avg. 28.7 Peptide Synthesis The synthesis ofa specific dipeptide, such as Ala-Gly from alanine and glycine, is complicated because both amino acids have two functional groups. As a result, four products—namely, Als Ala, Ala-Gly, Gly-Gly, and Gly-Ala—are possible. as Ey a Es naar Sea f s tet i! ay nae 8 fa & dion, a oy ala ‘i " 4H of Yate nav seo Son + He eon 8 fi fos, aircy ayaa How do we selectively join the COOH group of alanine with the NH, group of glycine? + Protect the functional groups that we don't want to react, and then form the amide bond.