Chapter 2 Section 3 PROTEINS
6. Formation of the Peptide Bond
The most important biological reaction of an amino acid is the formation of the peptide bond. Two amino acids can condense to form a special amide link between the α-carboxylic group of the first amino acid and the α-amino group of the second. This is the peptide bond, the fundamental covalent link responsible for building the backbone of a protein molecule.
+
H N H3
+ C C OO -
R' R''
H N H3
+ C C OO - +H + H O2
3 H N
R'
C N
H C
O H
R''
C C OO -
amino acid amino acid dipeptide
Fig 2.3.10. The formation of a peptide bond (shown enclosed in green) by condensation of two amino acids.
The peptide bond has a partial double bond character. It is a resonance hybrid of 2 structures in which the bond between C and N is single or double in nature. The resonance stabilization makes it stronger than a single bond.
The peptide bond is a dipole with its C-terminal end having a partial negative charge, and the N- terminal end having a partial positive charge.
C O-
N H N +
H C O
Fig 2.3.11a. The partial double bond nature of a peptide bond
Fig 2.3.11b. The dipole nature of a peptide bond.
Free rotation is not allowed between the carbonyl C (Co) of one amino acid and the α-amino N (Nα) of the next one in the chain i.e. the link between two amino acids is semi-rigid. Consequently the four atoms of the peptide bond viz. C, O, N and H are coplanar. However, free rotation can occur about the Cα and Nα bond (Ф-bond) and the Cα and CO (φ-bond).
A. B.
Fig 2.3.12. The character of the peptide bond: (A) shows its planar nature and (B) shows its dimensions (Source: (A) Berg et al, 2002, fig 3-23)
(B) Murray et al, 2003, p 20 fig 3-4)
The angles of the Ф and φ-bonds are known as Ramachandran angles; their degree of free rotation decides the conformation of the protein backbone.
2.3.2 PEPTIDES
Amino acids linked by peptide bonds constitute peptides. Oligopeptides contain less than 10 amino acids while polypeptides have upto 100 amino acids.
R
R
R C
N
N
C C
C N
C C
O O
1 O
2
3
CH2 N C O
C N
O CH
CH2 CH2 H
H COO-
NH H + 3
CH2 C
SH
COO-
A. A peptide chain B. γ-glutamyl-cysteinyl-glycine
Fig 2.3.13. A. General structure of a peptide chain (H atoms not shown) B. Structure of a tripeptide, glutathione
A peptide chain is a linear sequence of amino acids and is represented diagrammatically by a zig- zag line. The first amino acid has a free α-NH3+ group which, by convention, is written to the left.
The last amino acid has a free α-COO- group, written to the extreme right. Thus, the peptide/protein molecule has an amino-terminal end (N- terminal) and a carboxy-terminal end (C-terminal). The α-NH3+ and α-COO- groups of all other amino acyl residues are linked to form the peptide bonds that constitute the backbone of the peptide chain. The ‘R’ and ‘H’ groups project above and below the backbone and are in opposite directions in successive aminoacyl residues.
The linear sequence of amino acids represents the primary structure of the peptide and can be shown by using abbreviations for the constituents e.g. Glu-Ala-Lys-Gly-Tyr-Ala or E A K G Y A. If the order of the residues in any part of the chain is uncertain, the concerned amino acids are separated by commas and enclosed within brackets e.g. Glu-Lys-(Ala,Gly,Tyr,)-His-Ala
Most peptides have an assigned common name e.g. glutathione. For a more systematic nomenclature, the peptide is named as a derivative of the carboxy terminal amino acyl residue.
Each amino acyl residue in the sequence is named in order; with the –ine or -ate suffix replaced by –yl. Thus, glutathione is γ-glutamyl-cysteinyl-glycine (fig. 2.3.13.B)
Peptides have characteristic titration and isoelectric properties. The overall acid-base characters are due to the ‘R’ groups since the Co and Nα of all internal amino acids are covalently bonded.
The peptide bond can be cleaved by enzymatic or chemical hydrolysis. Carboxypeptidases release the C-terminal amino acids sequentially while aminopeptidases do the same for the N-terminal residues. Other enzymes like trypsin, chymotrypsin and pepsin hydrolyse more specific peptide links.
CLEAVAGE SITE H3
N+
COO- END
R CH2
C O C
O N+
H3 START N H
C H
N H
C H CH2 CH2 CH2
CLEAVAGE SITE
COO- END
R CH2
C O C
O +N
H3 START N H
C H
N H
C H
Trypsin acts on bond on C-side Chymotrypsin acts on bond on C-side of Lys/Arg residues of Phe/Tyr/Trp residues
Fig 2.3.14. Comparison of the specific peptide bonds hydrolyzed by the proteolytic enzymes trypsin and
Many peptides are of physiological importance. Thus, glutathione is a scavenger of harmful oxidizing agents in the body. Enkephalins are analgesic pentapeptides produced in the brain and nervous tissue. Bradykinin is anti-inflammatory. The posterior pituitary hormone oxytocin stimulates uterine contractions and milk ejection, while vasopressin has antidiuretic and vasopressor activities. The thyroid stimulating hormone, TSH, is a tripeptide. Insulin (51 amino acids) is the anti-diabetic hormone secreted by the pancreas.
N
Cys H N3+
Cys Tyr Ile
S
S Gln
Asn Pro Leu Gly C
O H2
Tyr Gly Gly Phe Leu
Oxytocin Leucine enkephalin
Insulin
Fig 2.3.15. Some physiologically important peptides.
2.3.3 PROTEINS
Polypeptides with more than 100 amino acids are called proteins. The different proteins differ in their constitution, configuration and conformation. These properties determine the plethora of complex functions that proteins perform in biological systems. The number of amino acyl residues in protein molecules varies considerably e.g. ubiquitin has 76 residues while titin has 27,000 residues.
The number of polypeptide chains in a protein also show considerable variations e.g. ribonuclease
= 1, insulin = 2, hemoglobin = 4, glutamine synthetase = 12. Monomeric proteins have a single continuous polypeptide chain, while oligomeric/multimeric proteins are constituted by two or more polypeptide chains/sub-units. Covalently-linked polypeptides in a protein are called chains and designated A, B, etc. Non-covalently linked polypeptides are called subunits and designated as α, β, γ etc. e.g. hemoglobin is a tetramer having 4, non-covalently bonded polypeptide chains.
It has 2 identical α-subunits and 2 identical β-subunits. Each α-subunit is paired with a β-subunit to give a structure of α2β2.
Fig 2.3.16. The α2β2 tetrameric structure of Hemoglobin. (Source: Berg et al, 2002, fig 3.49)
Grouping of proteins
Proteins may be grouped on the basis of different parameters:
• Composition: simple – having only amino acids (e.g. ribonuclease A), and conjugated – having additional non-protein groups (e.g. glycoprotein, lipoprotein)
• Shape: fibrous – having an axial ratio = 10 or more (e.g. collagen), and globular – with axial ratio not > 3 (e.g. enzymes)
• Function: structural (collagen), catalytic (enzymes), transport (hemoglobin), storage (ferritin), protection
(antibodies), endocrine (Growth hormone), receptors and growth factors General properties of proteins