Post on 22-Dec-2015
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
1
Chapter 26:Biomolecules: Amino Acids, Peptides, and Proteins
Based on McMurry’s Organic Chemistry, 6th edition©2003 Ronald KlugerDepartment of ChemistryUniversity of Toronto
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
2
Proteins – Amides from Amino Acids
Amino acids contain a basic amino group and an acidic carboxyl group
Joined as amides between the NH2 of one amino acid and the CO2H the next
Chains with fewer than 50 units are called peptides Protein: large chains that have structural or catalytic
functions in biology
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
3
26.1 Structures of Amino Acids
In neutral solution, the COOH is ionized and the NH2 is protonated
The resulting structures have “+” and “-” charges (a dipolar ion, or zwitterion)
They are like ionic salts in solution
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
4
The Common Amino Acids
20 amino acids form amides in proteins All are -amino acids - the amino and carboxyl are
connected to the same C They differ by the other substituent attached to the
carbon, called the side chain, with H as the fourth substituent except for proline
Proline, is a five-membered secondary amine, with N and the C part of a five-membered ring
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
5
Abbreviations and Codes
Alanine A, Ala
Arginine R, Arg
Asparagine N, Asn
Aspartic acid D, Asp
Cysteine C, Cys
Glutamine Q, Gln
Glutamic Acid E, Glu
Glycine G, Gly
Histidine H, His
Isoleucine I, Ile
Leucine L, Leu
Lysine K, Lys
Methionine M, Met
Phenylalanine F, Phe
Proline P, Pro
Serine S, Ser
Threonine T, Thr
Tryptophan W, Trp
Tyrosine Y, Tyr
Valine V, Val
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
6
Learning the Names and Codes
The names are not systematic so you learn them by using them (They become your friends)
One letter codes – learn them too If only one amino acid begins with that letter, use it (Cys,
His, Ile, Met, Ser, Val) If more than one begins with that letter, the more common
one uses the letter (Ala, Gly, Leu, Pro, Thr) For the others, some are phonetic: Fenylalanine, aRginine,
tYrosine Tryp has a double ring, hence W Amides have letters from the middle of the alphabet (Q –
Think of “Qtamine” for glutamine; asparagine -contains N “Acid” ends in D and E follows (smallest is first: aspartic
aciD, Glutamic acid E)
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
7
Neutral Hydrocarbon Side Chains
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
8
-OH, SH (Nucleophiles) and -S-CH3
Cysteine C, Cys
Methionine M, Met
Serine S, Ser
Threonine T, Thr
Tyrosine Y, Tyr
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
9
Acids and Amides
Aspartic acid D, Asp
Glutamic Acid E, Glu
Asparagine N, Asn
Glutamine Q, Gln
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
10
Amines
Arginine R, Arg
Histidine H, His
Lysine K, Lys
Tryptophan W, Trp
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
11
Chirality of Amino Acids
Glycine, 2-amino-acetic acid, is achiral In all the others, the carbons of the amino acids
are centers of chirality The stereochemical reference for amino acids is the
Fischer projection of L-serine Proteins are derived exclusively from L-amino acids
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
12
Types of side chains
Neutral: Fifteen of the twenty have neutral side chains
Asp and Glu have a second COOH and are acidic Lys, Arg, His have additional basic amino groups side
chains (the N in tryptophan is a very weak base) Cys, Ser, Tyr (OH and SH) are weak acids that are
good nucleophiles
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
13
Notes on Histidine
Contains an imidazole ring that is partially protonated in neutral solution
Only the pyridine-like, doubly bonded nitrogen in histidine is basic. The pyrrole-like singly bonded nitrogen is nonbasic because its lone pair of electrons is part of the 6 electron aromatic imidazole ring (see Section 24.4).
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
14
Essential Amino Acids
All 20 of the amino acids are necessary for protein synthesis
Humans can synthesize only 10 of the 20 The other 10 must be obtained from food
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
15
26.2 Isoelectric Points
In acidic solution, the carboxylate and amine are in their conjugate acid forms, an overall cation
In basic solution, the groups are in their base forms, an overall anion
In neutral solution cation and anion forms are present This pH where the overall charge is 0 is the
isoelectric point, pI
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
16
pI Depends on Side Chain
The 15 amino acids thiol, hydroxyl groups or pure hydrocarbon side chains have pI = 5.0 to 6.5 (average of the pKa’s)
D and E have acidic side chains and a lower pI H, R, K have basic side chains and higher pI
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
17
Electrophoresis
Proteins have an overall pI that depends on the net acidity/basicity of the side chains
The differences in pI can be used for separating proteins on a solid phase permeated with liquid
Different amino acids migrate at different rates, depending on their isoelectric points and on the pH of the aqueous buffer
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
18
Titration Curves of Amino Acids
If pKa values for an amino acid are known the fractions of each protonation state can be calculated (Henderson-Hasselbach Equation)
pH = pKa – log [A-]/[HA] This permits a titration curve to be calculated or pKa
to be determined from a titration curve
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
19
26.3 Synthesis of Amino Acids
Bromination of a carboxylic acid by treatment with Br2 and PBr3 (Section 22.4) then use NH3 or phthalimide (24.6) to displace Br
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
20
The Amidomalonate Synthesis
Based on malonic ester synthesis (see 22.8). Convert diethyl acetamidomalonate into enolate ion
with base, followed by alkylation with a primary alkyl halide
Hydrolysis of the amide protecting group and the esters and decarboxylation yields an -amino
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
21
Reductive Amination of -Keto Acids Reaction of an -keto acid with NH3 and a reducing
agent (see Section 24.6) produces an -amino acid
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
22
26.4 Enantioselective Synthesis of Amino Acids Amino acids (except glycine) are chiral and pure
enantiomers are required for any protein or peptide synthesis
Resolution of racemic mixtures is inherently ineffecient since at least half the material is discarded
An efficient alternative is enantioselective synthesis
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
23
Chemical Resolution of R,S Amino Acids Convert the amino group into an amide and react
with a chiral amine to form diastereomeric salts Salts are separated and converted back to the amino
acid by hydrolysis of the amide
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
24
Enzymic Resolution
Enzymes selectively catalyze the hydrolysis of amides formed from an L amino acid (S chirality center)
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
25
Enantioselective Synthesis of Amino Acids Chiral reaction catalyst creates diastereomeric
transition states that lead to an excess of one enantiomeric product
Hydrogenation of a Z enamido acid with a chiral hydrogenation catalyst produces S enantiomer selectively
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
26
26.5 Peptides and Proteins
Proteins and peptides are amino acid polymers in which the individual amino acid units, called residues, are linked together by amide bonds, or peptide bonds
An amino group from one residue forms an amide bond with the carboxyl of a second residue
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
27
Peptide Linkages
Two dipeptides can result from reaction between A and S, depending on which COOH reacts with which NH2 we get AS or SA
The long, repetitive sequence of NCHCO atoms that make up a continuous chain is called the protein’s backbone
Peptides are always written with the N-terminal amino acid (the one with the free NH2 group) on the left and the C-terminal amino acid (the one with the free CO2H group) on the right
Alanylserine is abbreviated Ala-Ser (or A-S), and serylalanine is abbreviated Ser-Ala (or S-A)
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
28
26.6 Covalent Bonding in Peptides
The amide bond that links different amino acids together in peptides is no different from any other amide bond (see Section 24.4). Amide nitrogens are nonbasic because their unshared electron pair is delocalized by interaction with the carbonyl group. This overlap of the nitrogen p orbital with the π orbitals 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 therefore planar, and the N–H is oriented 180° to the C=O.
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
29
26.6 Covalent Bonding in Peptides
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
30
Disulfides
Thiols in adjacent chains can form a disulfide RS–SR through spontaneous oxidation (see 18.10)
A disulfide bond between cysteine residues in different peptide chains links the otherwise separate chains together, while a disulfide bond between cysteine residues in the same chain forms a loop
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
31
26.7 Structure Determination of Peptides: Amino Acid Analysis The sequence of amino acids in a pure protein is
specified genetically If a protein is isolated it can be analyzed for its
sequence The composition of amino acids can be obtained by
automated chromatography and quantitative measurement of eluted materials using a reaction with ninhydrin that produces an intense purple color
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
32
Amino Acid Analysis Chromatogram
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
33
26.8 Peptide Sequencing: The Edman Degradation The Edman degradation cleaves amino acids one at
a time from the N-terminus and forms a detectable, separable derivative for each amino acid
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
34
26.9 Peptide Sequencing: C-Terminal Residue Determination Carboxypeptidase enzymes cleave the C-terminal
amide bond Analysis determines the appearance of the first free
amino acid, which must be at the carboxy terminus of the peptide
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
35
26.10 Peptide Synthesis
Peptide synthesis requires that different amide bonds must be formed in a desired sequence
The growing chain is protected at the carboxyl terminal and added amino acids are N-protected
After peptide bond formation, N-protection is removed
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
36
Carboxyl Protecting Groups
Usually converted into methyl or benzyl esters Removed by mild hydrolysis with aqueous NaOH Benzyl esters are cleaved by catalytic hydrogenolysis
of the weak benzylic C–O bond
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
37
Amino Group Protection
An amide that is less stable than the protein amide is formed and then removed
The tert-butoxycarbonyl amide (BOC) protecting group is introduced with di-tert-butyl dicarbonate
Removed by brief treatment with trifluoroacetic acid
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
38
Peptide Coupling
Amides are formed by treating a mixture of an acid and amine with dicyclohexylcarbodiimide (DCC)
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
39
Overall Steps in Peptide Synthesis
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
40
26.11 Automated Peptide Synthesis: The Merrifield Solid-Phase Technique
Peptides are connected to beads of polystyrene, reacted, cycled and cleaved at the end
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
41
Automated Synthesis
The solid-phase technique has been automated, and computer-controlled peptide synthesizers are available for automatically repeating the coupling and deprotection steps with different amino acids
Applied Biosystems® Synthesizer
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
42
26.12 Protein Classification
Simple proteins yield only amino acids on hydrolysis Conjugated proteins, which are much more common
than simple proteins, yield other compounds such as carbohydrates, fats, or nucleic acids in addition to amino acids on hydrolysis.
Fibrous proteins consist of polypeptide chains arranged side by side in long filaments
Globular proteins are coiled into compact, roughly spherical shapes
Most enzymes are globular proteins
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
43
Some Common Fibrous and Globular Proteins
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
44
26.13 Protein Structure
The primary structure of a protein is simply the amino acid sequence.
The secondary structure of a protein describes how segments of the peptide backbone orient into a regular pattern.
The tertiary structure describes how the entire protein molecule coils into an overall three-dimensional shape.
The quaternary structure describes how different protein molecules come together to yield large aggregate structures
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
45
-Keratin
A fibrous structural protein coiled into a right-handed helical secondary structure, -helix stabilized by H-bondsb between amide N–H groups and C=O groups four residues away a-helical segments in their chains
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
46
Fibroin
Fibroin has a secondary structure called a -pleated sheet in which polypeptide chains line up in a parallel arrangement held together by hydrogen bonds between chains
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
47
Myoglobin
Myoglobin is a small globular protein containing 153 amino acid residues in a single chain
8 helical segments connected by bends to form a compact, nearly spherical, tertiary structure
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
48
Internal and External Forces
Acidic or basic amino acids with charged side chains congregate on the exterior of the protein where they can be solvated by water
Amino acids with neutral, nonpolar side chains congregate on the hydrocarbon-like interior of a protein molecule
Also important for stabilizing a protein's tertiary structure are the formation of disulfide bridges between cysteine residues, the formation of hydrogen bonds between nearby amino acid residues, and the development of ionic attractions, called salt bridges, between positively and negatively charged sites on various amino acid side chains within the protein
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
49
26.14 Enzymes
An enzyme is a protein that acts as a catalyst for a biological reaction.
Most enzymes are specific for substrates while enzymes involved in digestion such as papain attack many substrates
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
50
Cofactors
In addition to the protein part, many enzymes also have a nonprotein part called a cofactor
The protein part in such an enzyme is called an apoenzyme, and the combination of apoenzyme plus cofactor is called a holoenzyme. Only holoenzymes have biological activity; neither cofactor nor apoenzyme can catalyze reactions by themselves
A cofactor can be either an inorganic ion or an organic molecule, called a coenzyme
Many coenzymes are derived from vitamins, organic molecules that are dietary requirements for metabolism and/or growth
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
51
Types of Enzymes by Function
Enzymes are usually grouped according to the kind of reaction they catalyze, not by their structures
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
52
26.15 How Do Enzymes Work? Citrate Synthase Citrate synthase catalyzes a mixed Claisen
condensation of acetyl CoA and oxaloacetate to give citrate
Normally Claisen condensation require a strong base in an alcohol solvent but citrate synthetase operates in neutral solution
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
53
The Structure of Citrate Synthase
Determined by X-ray crystallography Enzyme is very large compared to substrates,
creating a complete environment for the reaction
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
54
Mechanism of Citrate Synthetase
A cleft with functional groups binds oxaloacetate Another cleft opens for acetyl CoA with H 274 and D
375, whose carboxylate abstracts a proton from acetyl CoA
The enolate (stabilized by a cation) adds to the carbonyl group of oxaloacetate
The thiol ester in citryl CoA is hydrolyzed
Based on McMurry, Organic Chemistry, Chapter 26, 6th edition, (c) 2003
55
26.16 Protein Denaturation
The tertiary structure of a globular protein is the result of many intramolecular attractions that can be disrupted by a change of the environment, causing the protein to become denatured
Solubility is drastically decreased as in heating egg white, where the albumins unfold and coagulate
Enzymes also lose all catalytic activity when denatured