Amino Acids and Proteins Created By Prof Gary

1. Introduction • Amino acid: a compound that contains both an amino group and a carboxyl group • Amino acids are the monomeric unit for proteins. © 2014 by John Wiley & Sons, Inc. All rights reserved. 2

1. Introduction There are 20 different α amino acids, and 4 levels of protein

2. Amino Acids • In neutral solution, the COOH is ionized and the NH 2 is protonated • The resulting structures have “+” and “-” charges (a dipolar ion, or zwitterion) German zwitter, meaning “hybrid” • They are like ionic salts in solution Dipolar ion p. H 6 © 2014 by John Wiley & Sons, Inc. All rights reserved. Cationic form p. Ka 1= 2. 3 4 Anionic form p. Ka 2= 9. 7

© 2014 by John Wiley & Sons, Inc. All rights reserved. 5

2. Amino Acids 2 A. Structures and Names The four classes of amino acids are: Neutral, Nonpolar Neutral, Polar Charged Positive, Polar Acidic (at p. H 7) Charged Negative, Polar Basic (at p. H 7) © 2014 by John Wiley & Sons, Inc. All rights reserved. 6

2. Amino Acids Structures of hydrophobic amino acids © 2014 by John Wiley &

2. Amino Acids Structures of the polar amino acids © 2014 by John Wiley

2. Amino Acids Negatively charged amino acids Positively charged amino acids © 2014 by

2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

2. Amino Acids • Note the titration curve for Alanine below. – As base is added and p. H is increased, the net charge on Alanine goes from positive to negative. Dipolar ions are also called zwitterions. Isoelectric point (p. I) is the p. H at which the net charge on an amino acid is 0. © 2014 by John Wiley & Sons, Inc. All rights reserved. 12

2. Amino Acids Glutamic acid Practice Problem 24. 1 What form of glutamic acid would you expect to predominate in (a) strongly acidic solution, (b) strongly basic solution? © 2014 by John Wiley & Sons, Inc. All rights reserved. 13

3. Synthesis of α–Amino Acids 3 B. The Strecker Synthesis • treat aldehyde with NH 3 and HCN, and • produce an α amino acid. 14

3. Synthesis of α–Amino Acids Practice Problem 24. 4 (a) Outline a Strecker synthesis of dl-phenylalanine. by a Strecker synthesis. The required starting aldehyde can be prepared from acrolein © 2014 by John Wiley & Sons, Inc. All rights reserved. 15

4. Polypeptides and Proteins • Peptides or protein= – are chain of amino acids Amino acid residues connected by peptide bonds • Residue = AA – H 2 O No. of Amino Acids linked together Name 2 Dipeptide 3 Tripeptide 3 -10 Oligopeptide Multiple polypeptides Protein C terminus on right N terminus on left Amide bond or peptide bond © 2014 by John Wiley & Sons, Inc. All rights reserved. 16

Resonance Structures of Peptide Bond The four atoms of a peptide bond and the two alpha carbons joined to it lie in a plane with bond angles of 120°about C and N to account for this geometry, a peptide bond is most accurately represented as a hybrid of two contributing structures (resonance structures) • the hybrid has considerable C-N double bond character and rotation about the peptide bond is restricted - Amide nitrogens are nonbasic because their unshared electron pair is delocalized by interaction with the carbonyl group - The overlap of the nitrogen p orbital with the p orbital of the carbonyl group imparts a certain amount of double-bond character to the C-N bond and restricts rotation around it - The amide bond is planar - The N-H is oriented 180º to the C=O © 2014 by John Wiley & Sons, Inc. All rights reserved. 17

4. Polypeptides and Proteins 4 A. Hydrolysis • Peptide bond cleavage occurs in 6 M HCl for 24 h. • AAs separate on cation-exchange resin. – AAs will bind at low p. H. Acids (AA) – AAsisrelease as p. H increases. the eluate allowed to mix with Resin Ninhydrin, a reagent that reacts with most amino acids to give a derivative with an intense purple color (λmax 570 nm). The amino acid analyzer is designed so that it can measure the absorbance of the eluate (at 570 nm) continuously and record this absorbance as a function of the © 2014 by John Wiley & Sons, Inc. All rights reserved. 18 Amino

4. Polypeptides and Proteins Detect AAs Automatic Amino Acid Analyzer – Ninhydrin Reagent – λmax = 570 nm Formation of blue complex occurs with this reagent. Exception of proline and hydroxyproline α-amino acids notice that the only portion of the anion that is derived from the 19 a-amino acid is the

5. Primary Structure of Polypeptides and Proteins Primary Structure is defined by the – molecular weight, – amino acid composition, and – amino acid sequence. Edman Degradation Involves – identification of N-Terminus, – sequential degradation of AAs from N- terminus, – And is good up to 60 AAs. © 2014 by John Wiley & Sons, Inc. All rights reserved. 20

5. Primary Structure of Polypeptides and Proteins 5 A. Edman Degradation cleaved from the peptide chain by acid Procedure for identifying the Nterminal amino acid in a peptide labeling reaction between the N-terminal amino group and Phenyl isothiocyanate Identification by HPLC with known amino acid Anilinothioazolinone (ATZ) © 2014 by John Wiley & Sons, Inc. All rights reserved. 21

5. Primary Structure of Polypeptides and Proteins Nucleophilic aromatic substitution reaction 5 B. Sanger N-Terminus Analysis involves – sequential Analysis, and – use of a 2, 4 -dinitroflourobenzene dye. © 2014 by John Wiley & Sons, Inc. All rights reserved. 22

The electron-withdrawing property of the 2, 4 -dinitrophenyl group makes separation of the labeled amino acid very easy. Suggest how this is done. © 2014 by John Wiley & Sons, Inc. All rights reserved. 23

• Complete Sequence Analysis Sequential analysis using the Edman degradation or other methods becomes impractical with large proteins and polypeptides. • Partial hydrolysis with dilute acid, for example, generates a family of peptides cleaved in random locations and with varying lengths. Sequencing these cleavage peptides and looking for points of overlap allows the sequence of the entire peptide to be pieced together • Example: We are given a pentapeptide known to contain valine (two residues), leucine (one residue), histidine (one residue), and phenylalanine (one residue). Ø write “Molecular formula” of the protein in the following way, using commas to indicate that the sequence is unknown: 2 V, L, H, F by using DNFB and carboxypeptidase. If Valine and leucine are the N- and C-terminal residues, respectively. So far we know the following: V (V, H, F) L - sequence of the three nonterminal amino acids (V, H, F) is still unknown. - subject the pentapeptide to partial acid hydrolysis and obtain the following dipeptides: VH + HV + VF + FL The points of overlap of the dipeptides (i. e. , H, V, and F) tell us that the original pentapeptide VH must have been the following: VHVFL HV VF FL 24 © 2014 by John Wiley & Sons, Inc. All rights reserved. VHVFL

5. Primary Structure of Polypeptides and Proteins 5 D. Complete Sequence Analysis – starts with C & N Terminus Identification – followed by fragment analysis Practice Problem 24. 9 © 2014 by John Wiley & Sons, Inc. All rights reserved. 25

Glutathione is a tripeptide found in most living cells. Partial acid-catalyzed hydrolysis of glutathione yields two dipeptides, CG and one composed of E and C. When this second dipeptide was treated with DNFB, acid hydrolysis gave N-labeled glutamic acid. (a) On the basis of this information alone, what structures are possible for glutathione? is theexperiments structure of have glutathione? b)What Synthetic shown that the second dipeptide has the following structure: © 2014 by John Wiley & Sons, Inc. All rights reserved. 26

5. Primary Structure of Polypeptides and Proteins 5 E. Peptide Sequencing Use Mass Spectrometry and Sequence Databases – Ladder Sequence • uses carboxypeptidases, • analyzes the molecule weight of the polypeptides (PPs) which have been degraded, and • each PP is one AA larger than the last. – Tandem Mass Spectrometry • analyzes fragments generated by MS. – Partial Hydrolysis and Sequence Comparison • is used for analyzing small chains within known PP databases, and • identification of a known PP is possible. © 2014 by John Wiley & Sons, Inc. All rights reserved. 27

6. Examples of Polypeptide and Protein Primary Structure The covalent structure of a protein

6. Examples of Polypeptide and Protein Primary Structure Oxytocin and Vasopressin • Oxytocin stimulates uterine contractions during childbirth. • Vasopressin stimulates peripheral blood vessel contraction. The structures of oxytocin and vasopressin also illustrate the importance of the disulfide linkage between cysteine residues in the overall primary structure of a polypeptide. In these two molecules this disulfide linkage leads to a cyclic structure. © 2014 by John Wiley & Sons, Inc. All rights reserved. 29

6. Examples of Polypeptide and Protein Primary Structure 6 B. Insulin – is secreted by pancreas, and – regulates (lowers) blood glucose. Thousands of other polypeptides and protein structures are known. © 2014 by John Wiley & Sons, Inc. All rights reserved. 30

7. Polypeptide and Protein Synthesis Protecting Groups • Used to prevent C-terminus from reacting with N-Terminus. • Examples of N-Terminus protecting groups are below. © 2014 by John Wiley & Sons, Inc. All rights reserved. 31

7. Polypeptide and Protein Synthesis Z group Benzyl carbocation The easy removal of the Z group in acidic media results from the exceptional stability of the carbocations that are formed initially. The benzyloxycarbonyl group gives a benzyl carbocation. Removal of the benzyloxycarbonyl group with hydrogen and a catalyst depends on the fact that benzyl–oxygen bonds are weak and subject to hydrogenolysis at low temperatures, resulting in methylbenzene (toluene) as one product © 2014 by John Wiley & Sons, Inc. All rights reserved. 32

7. Polypeptide and Protein Synthesis 7 B. Activation of the Carboxyl Group • is needed to get the C-Terminus to react, • To convert the carboxyl group of the “protected” amino acid to a mixed anhydride using ethyl chloroformate Z group The mixed anhydride can then be used to acylate another amino acid and form a peptide linkage: © 2014 by John Wiley & Sons, Inc. All rights reserved. 33

7. Polypeptide and Protein Synthesis 7 C. Peptide Synthesis Involves several steps: –N-Terminus Protection –C-Terminus Activation –Amino Acid Addition Let us examine now how we might use these reagents in the preparation of the simple dipeptide AL © 2014 by John Wiley & Sons, Inc. All rights reserved. 34

8. Secondary, Tertiary, and Quaternary Structures of Proteins Secondary Structure • is defined by local conformation of polypeptides. • Specific types are – α helix, – β sheets, and – coil or loop. • Rotation – AA have limited or no rotation. – The side groups have free rotation. – There is free rotation about the Peptide bond. © 2014 by John Wiley & Sons, Inc. All rights reserved. 35

• A transoid arrangement of groups around the relatively rigid amide bond would cause the side-chain R groups to alternate from side to side of a single fully extended peptide chain: Calculations show that such a polypeptide chain would have a repeat distance (i. e. , distance between alternating units) of 7. 2 Å © 2014 by John Wiley & Sons, Inc. All rights reserved. 36

8. Secondary, Tertiary, and Quaternary Structures of Proteins Secondary Structure H- Bond • β- pleated sheet (below) has – high nonpolar AA content, and – predominates in silk. The predominant secondary structure in silk fibroin (48% glycine and 38% serine and alanine residues) is the β -pleated sheet. H- Bond • α helix (right) has – 3. 6 AA’s per right handed turn, and – is in fibrous proteins. © 2014 by John Wiley & Sons, Inc. All rights reserved. 37

8. Secondary, Tertiary, and Quaternary Structures of Proteins 8 B. Tertiary Structure – has a three dimensional shape that arises from secondary structures orientation to each other, and – results in two classes of proteins. • Fibrous Proteins have mainly α helices. • Globular Proteins – have all types of secondary structure. – AA classes have specific locations: • nonpolar in the interior and • polar in the exterior. © 2014 by John Wiley & Sons, Inc. All rights reserved. 38

8. Secondary, Tertiary, and Quaternary Structures of Proteins Carbonic Anhydrase Myoglobin © 2014 by

8. Secondary, Tertiary, and Quaternary Structures of Proteins 8 C. Quaternary Structures Three dimensional shape that arises from polypeptide chain orientation to each other. • See the four polypeptide chains of hemoglobin to the right. © 2014 by John Wiley & Sons, Inc. All rights reserved. 40

9. Introduction to Enzymes • Enzymes – catalyze all chemical reactions found in cellular metabolism, – have highly specific reactants called substrates, – form an enzyme-substrate complex, and – are stereospecific. • Enzyme-substrate complex – induces conformational changes in the enzyme, – is the place where the reaction is catalyzed, and – resides within the active site. © 2014 by John Wiley & Sons, Inc. All rights reserved. 41

• Enzymes show remarkable specificity for their substrates and formation of specific products. For example: Polypeptides consisting of well over 1000 amino acid residues are synthesized virtually without error. Emil Fischer’s in 1894: The ability of enzymes to distinguish between a- and b-glycosidic linkages. Lid him to formulate his lock-and-key hypothesis for enzyme specificity. a lock-and-key hypothesis, the specificity of an enzyme (the lock) and its substrate (the key) comes from their geometrically complementary shapes. a In an enzyme-catalyzed reaction, the enzyme and the substrate combine to form an enzyme–substrate complex. a Formation of the enzyme–substrate complex often induces a conformational change in the enzyme called an induced fit that allows it to bind the substrate more effectively. s O Ser Phe s H CO 2 Asp The place where a substrate binds to an enzyme and where the reaction takes place is 42 called the active site. © 2014 by John Wiley & Sons, Inc. All rights reserved.

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9. Introduction to Enzymes • Competitive Inhibitors – have a similar structure to substrate and – binds to the active site instead of the substrate. • Cofactors – is an extra molecule required for enzyme activity. – i. e. metal ions • Coenzyme – Organic cofactor © 2014 by John Wiley & Sons, Inc. All rights reserved. 44

Formulated as Hydrochloride Salt Side chain is ionized and negatively charged Rimantidine (racemic mixture)

10. Lysozyme: Mode of Action of an Enzyme • Lysozymes cleave the cell wall of gram positive bacteria. • Lysozymes hydrolyze the glycosidic linkages in peptoglycans. • Blue AAs are found at the active site. © 2014 by John Wiley & Sons, Inc. All rights reserved. 46

Lysozyme digests bacterial cell walls by breaking b(1 - 4) glycosidic bonds between (N-

10. Lysozyme: Mode of Action of an Enzyme Below is the substrate that Lysozyme

10. Lysozyme: Mode of Action of an Enzyme Below is the AA in Lysozyme

12. Hemoglobin: A Conjugated Protein • Hemoglobin is a conjugated protein with a nonprotein prosthetic group. • heme is the prosthetic group. • Fe 2+ © 2014 by John Wiley & Sons, Inc. All rights reserved. 50

The iron of the heme group forms a coordinate bond to a nitrogen of the imidazole group of histidine of the polypeptide chain. This leaves one valence of the ferrous ion free to combine with oxygen as follows: © 2014 by John Wiley & Sons, Inc. All rights reserved. 51

• When the heme combines with oxygen the ferrous ion (Fe 2+) does not become readily oxidized to the ferric state. ((why? ? )) • Studies with model heme compounds in water, for example, show that they undergo a rapid combination with oxygen but they also undergo a rapid oxidation of the iron from Fe 2+ to Fe 3+. When these same compounds are embedded in the hydrophobic environment of a polystyrene resin, however, the iron is easily oxygenated and deoxygenated, and this occurs with no change in oxidation state of iron. note that X-ray studies of hemoglobin have revealed that the polypeptide chains provide each heme group with a similar hydrophobic environment. © 2014 by John Wiley & Sons, Inc. All rights reserved. 52

13. Purification and Analysis of Polypeptides and Proteins 13 B. Analysis Methods – Gel Electrophoresis (based upon p. I) – Mass Spectroscopy • Obtains molecular weight – Electrospray Ionization (ESI) uses • peak corresponds to m/z ratio and • mixtures separated by HPLC – Matrix-assisted Laser Desorption Ionization (MALDI) • Used for ionization of nonvolatile molecules © 2014 by John Wiley & Sons, Inc. All rights reserved. 53