Prentice Hall c2002Chapter 31 Principles of Biochemistry Fourth Edition Chapter 3 Amino Acids and...

Prentice Hall c2002 Chapter 3 1

Principles of BiochemistryFourth Edition

Chapter 3Amino Acids and the Primary

Structures of Proteins

Copyright © 2006 Pearson Prentice Hall, Inc.

Horton • Moran • Scrimgeour • Perry • Rawn


Chapter 3 - Amino Acids and the Primary Stucture of Proteins

Important biological functions of proteins

1. Enzymes, the biochemical catalysts

2. Storage and transport of biochemical molecules

3. Physical cell support and shape (tubulin, actin, collagen)

4. Mechanical movement (flagella, mitosis, muscles)

(continued)


Functions of proteins (continued)

5. Decoding cell information (translation, regulation of gene expression)

6. Hormones or hormone receptors (regulation of cellular processes)

7. Other specialized functions (antibodies, toxins etc)


3.1 General Structure of Amino Acids

• Twenty common -amino acids have carboxyl and amino groups bonded to the -carbon atom

• A hydrogen atom and a side chain (R) are also attached to the -carbon atom


Zwitterionic form of amino acids

• Under normal cellular conditions amino acids are zwitterions (dipolar ions):

Amino group = -NH3+

Carboxyl group = -COO-


Fig 3.1 Two representations of an amino acid at neutral pH

(b) Ball-and stick model

(a) Structure


Stereochemistry

• Stereoisomers (立體異構物 )- compounds that have the same molecular formula but differ in the arrangement of atoms in space

• Enantiomers - nonsuperimposable mirror images

• Chiral carbons - have four different groups attached


Stereochemistry of amino acids

• 19 of the 20 common amino acids have a chiral -carbon atom (Gly does not)

• Threonine and isoleucine have 2 chiral carbons each (4 possible stereoisomers each)

• Mirror image pairs of amino acids are designated L (levo) and D (dextro)

• Proteins are assembled from L-amino acids (a few D-amino acids occur in nature)


Fig 3.2 Mirror-image pairs of amino acids


Assignment of configuration by the RS system

(a) Assign a priority to each group attached to a chiral carbon based upon atomic mass priority (1 highest, 4 lowest)

• If two atoms are identical, move to the next atoms

• For double or triple bonds, count atom once for each bond (-CHO higher priority than -CH2OH)

• Priorities (low to high): -H, -CH3, -C6H5, -CH2OH,-CHO, -COOH, -NH2, -NHR, -OH, -OR, -SH


RS system (continued)

(b) Orient the molecule with priority 4 pointing away (behind the chiral carbon). Trace path from highest priority to lowest priority (1, 2, 3, 4)

(c) Clockwise path: absolute configuration R

Counterclockwise path: absolute configuration S

NOTE: All of the 19 common chiral L-amino acids except cysteine have the S configuration.


Assignment of RS configuration


3.2 Structures of the 20 Common Amino Acids

• Fischer projections - horizontal bonds from a chiral center extend toward the viewer, vertical bonds extend away from the viewer

• Abbreviations can be one letter or three letters

• Amino acids are grouped by the properties of their side chains (R groups)

• Classes: Aliphatic, Aromatic, Sulfur-containing, Alcohols, Bases, Acids and Amides


A. Aliphatic R Groups

• Glycine (Gly, G) - the -carbon is not chiral

since there are two H’s attached (R=H)

• Four amino acids have saturated side chains:

Alanine (Ala, A) Valine (Val, V)

Leucine (Leu, L) Isoleucine (Ile, I)

• Proline (Pro, P) 3-carbon chain connects -C and N


Four aliphatic amino acid structures


Fig 3.3 Stereoisomers of Isoleucine

• Ile has 2 chiral carbons, 4 possible stereoisomers


Proline has a nitrogen in the aliphatic ring system

• Proline (Pro, P) - has a three carbon side chain bonded to the -amino nitrogen

• The heterocyclic pyrrolidine ring restricts the geometry of polypeptides


B. Aromatic R Groups

• Side chains have aromatic groups

Phenylalanine (Phe, F) - benzene ring

Tyrosine (Tyr, Y) - phenol ring

Tryptophan (Trp, W) - bicyclic indole group


Aromatic amino acid structures


C. Sulfur-Containing R Groups

• Methionine (Met, M) - (-CH2CH2SCH3)

• Cysteine (Cys, C) - (-CH2SH)

• Two cysteine side chains can be cross-linked by forming a disulfide bridge (-CH2-S-S-CH2-)

• Disulfide bridges may stabilize the three-dimensional structures of proteins


Methionine and cysteine


Fig 3.4 Formation of cystine


D. Side Chains with Alcohol Groups

• Serine (Ser, S) and Threonine (Thr, T) have uncharged polar side chains


E. Basic R Groups

• Histidine (His, R) - imidazole

• Lysine (Lys, K) - alkylamino group

• Arginine (Arg, R) - guanidino group

• Side chains are nitrogenous bases which are substantially positively charged at pH 7


Structures of histidine, lysine and arginine


F. Acidic R Groups and Amide Derivatives

• Aspartate (Asp, D) and Glutamate (Glu, E) are dicarboxylic acids, and are negatively charged at pH 7

• Asparagine (Asn, N) and Glutamine (Gln, Q) are uncharged but highly polar


Structures of aspartate, glutamate, asparagine and glutamine


G. The Hydrophobicity of Amino Acid Side Chains

• Hydropathy: the relative hydrophobicity of each amino acid

• The larger the hydropathy, the greater the tendency of an amino acid to prefer a hydrophobic environment

• Hydropathy affects protein folding: hydrophobic side chains tend to be in the interiorhydrophilic residues tend to be on the surface


Table 3.1

• Hydropathy scale for amino acid residues

(Free-energy change for transfer of an amino acid from interior of a lipid bilayer to water)

Free-energy change for transfer (kjmol-1)

Aminoacid


3.3 Other Amino Acids and Amino Acid Derivatives

• Over 200 different amino acids are found in nature

• Most are precursors to common amino acids or chemically modified derivatives

• Some amino acids are chemically modified after incorporation into a polypeptide


Fig 3.5 Compounds derived from common amino acids


Selenocysteine

• Selenocysteine is incorporated into a few proteins

• Constitutes the 21st

amino acid


3.4 Ionization of Amino Acids

• Ionizable groups in amino acids: (1) -carboxyl, (2) -amino, (3) some side chains

• Each ionizable group has a specific pKa

AH B + H+

• For a solution pH below the pKa, the protonated form predominates (AH)

• For a solution pH above the pKa, the unprotonated form predominates (B)


Fig 3.6 Titration curve for alanine

• Titration curves are used to determine pKa values

• pK1 = 2.4

• pK2 = 9.9

• pIAla = isoelectric point


Fig 3.7 Ionization of Histidine

(a) Titration curve of histidine

pK1 = 1.8pK2 = 6.0pK3 = 9.3


Fig 3.7 (b) Deprotonation of imidazolium ring


Table 3.2

pKa values of amino acid ionizable groups


Henderson-Hasselbach equation: calculating group ionizations

[proton acceptor]

pH = pKa + log

[proton donor]


Fig 3.8 (a) Ionization of the protonated -carboxyl of glutamate


Fig 3.8 (b) Deprotonation of the guanidinium group of Arg


3.5 Peptide Bonds Link Amino Acids in Proteins

• Peptide bond - linkage between amino acids is a secondary amide bond

• Formed by condensation of the -carboxyl of one amino acid with the -amino of another amino acid (loss of H2O molecule)

• Primary structure - linear sequence of amino acids in a polypeptide or protein


Fig 3.9 Peptide bond between two amino acids


Polypeptide chain nomenclature

• Amino acid “residues” compose peptide chains

• Peptide chains are numbered from the N (amino) terminus to the C (carboxyl) terminus

• Example: (N) Gly-Arg-Phe-Ala-Lys (C) (or GRFAK)

• Formation of peptide bonds eliminates the ionizable -carboxyl and -amino groups of the free amino acids


Fig 3.10 Aspartame, an artificial sweetener

• Aspartame is a dipeptide methyl ester (aspartylphenylalanine methyl ester)

• About 200 times sweeter than table sugar

• Used in diet drinks


3.6 Protein Purification Techniques

• Common types of column chromatography:

Ion-exchange chromatography - separation based upon the overall charge of molecules

Gel-filtration chromatography - separation based upon molecular size

Affinity chromatography - separation by specific binding interactions between column matrix and target proteins


Fig 3.11 Column Chromatography

(a) Separation of a protein mixture

(b) Detection of eluting protein peaks


Electrophoresis

• Polyacrylamide gel electrophoresis (PAGE) Separates molecules on a polyacrylamide gel matrix when an electric field is applied

• SDS-PAGE. Sodium dodecyl sulfate (SDS) coats proteins with negative charges. Coated polypeptide chains then separate by molecular mass (method to determine molecular weight)


Fig 3.12 (a) SDS-PAGE Electrophoresis (b) Protein banding pattern after run


Fig. 3.14


3.7 Amino Acid Composition of Proteins

• Amino acid analysis - determination of the amino acid composition of a protein

• Peptide bonds are cleaved by acid hydrolysis (6M HCl, 110o, 16-72 hours)

• Amino acids are separated chromatographically and quantitated

• Phenylisothiocyanate (PITC) used to derivatize the amino acids prior to HPLC analysis


Fig 3.15 Acid-catalyzed hydrolysis of a peptide


Fig 3.16 Amino acid treated with PITC


Fig 3.17 Chromatogram from HPLC-separated PTC-amino acids


3.8 Determining the Sequence of Amino Acids

• Edman degradation procedure - Determining one residue at a time from the N-terminus

(1) Treat peptide with PITC which reacts with the N-terminus to form a PTC-peptide

(2) Treat with trifluoroacetic acid (TFA) to selectively cleave the N-terminal peptide

bond

(3) Separate N-terminal derivative from peptide

(4) Convert derivative to PTH-amino acid


Fig 3.18 Edman degradation procedure

Phenylisothiocyanate(Edman reagent)

pH = 9.0

Phenylthiocarbamoyl-peptide

F3CCOOH


Edman degradation procedure (cont)

Aqueous acid

Polypeptide chain with n-1 amino acids

Returned to alkaline conditionsfor reaction with additional phenylisothiocyanate in the next cycle of Edman degradation


Cleaving and blocking disulfide bonds

• Disulfide bonds in proteins must be cleaved:

(1) To permit isolation of the PTH-cysteine during the Edman procedure

(2) To separate peptide chains

• Treatment with thiol compounds reduces the (R-S-S-R) cystine bond to two cysteine (R-SH) residues


Fig 3.19 Cleaving, blocking disulfide bonds


3.9 Protein Sequencing Strategies

• Proteins may be too large to be sequenced completely by the Edman method

• Proteases (enzymes cleaving peptide bonds) and chemical agents are used to selectively cleave the protein into smaller fragments

• Cyanogen bromide (BrCN) cleaves polypeptides at the C-terminus of Met residues


Fig 3.20 Protein cleavage by BrCN


Protease enzymes cleave specific peptide bonds

• Chymotrypsin - carbonyl side of aromatic or bulky noncharged aliphatic residues (e.g. Phe, Tyr, Trp, Leu)

• Trypsin - carbonyl side, basic residues (Lys,Arg).

• Staphylococcus aureus V8 protease - carbonyl side of negatively charged residues (Glu, Asp). NOTE: in 50mM ammonium bicarbonate cleaves only at Glu.


Fig 3.21 Cleavage, sequencing an oligopeptide


Fig 3.20 Sequences of DNA and protein

• Protein amino acid sequences can be deduced from the sequence of nucleotides in the corresponding gene

• A sequence of three nucleotides specifies one amino acid (A,C,G,T are DNA residues )


3.10 Comparisons of the Primary Structures of Proteins Reveal Evolutionary Relationships

• Closely related species contain proteins with very similar amino acid sequences

• Differences reflect evolutionary change from a common ancestral protein sequence

• Cytochrome c protein sequences from various species can be aligned to show their similarities

• Phylogenetic tree shows evolutionary differences in amino acid sequences


Fig. 3.23


Fig 3.24

Phylogenetic tree for cytochrome c

Prentice Hall c2002Chapter 31 Principles of Biochemistry Fourth Edition Chapter 3 Amino Acids and...

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