1. Amino Acids, Peptides, and Proteins
Dr. Wolf's CHM 424 4

2. Objectives
Draw a general amino acid and identify the two functional groups common to all.
Classify each amino acid according to the chemical nature of its R group.
Define the meaning of an essential amino acid.
Draw the reaction that joins two amino acids to form a peptide bond.
Describe and differentiate primary, secondary, tertiary, and quaternary protein structures.
Describe and differentiate co-enzymes and prosthetic groups.
List and discuss four forces that stabilize globular protein structure.
List important structural similarities and differences between myoglobin and hemoglobin.
Describe the mutation present in hemoglobin giving rise to sickle cell disease.
Dr. Wolf's CHM 424

3. Classification of Amino Acids
Dr. Wolf's CHM 424 4

4. Fundamentals
While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively.
They are classified as , , , etc. amino acids according the carbon that bears the nitrogen.
Dr. Wolf's CHM 424

5. Amino Acids + NH3  – CO2 + – H3NCH2CH2CO2  + – H3NCH2CH2CH2CO2 
an -amino acid that is an intermediate in the biosynthesis of ethylene 
a -amino acid that is one of the structural units present in coenzyme A +
H3NCH2CH2CO2 –  +
H3NCH2CH2CH2CO2 –
a -amino acid involved in the transmission of nerve impulses 
Dr. Wolf's CHM 424

6. They differ in respect to the group attached to the  carbon.
The 20 Key Amino Acids
More than 700 amino acids occur naturally, but 20 of them are especially important.
These 20 amino acids are the building blocks of proteins. All are -amino acids.
They differ in respect to the group attached to the  carbon.
These 20 are listed in Table 27.1.
Dr. Wolf's CHM 424

7. The properties of the amino acid vary as the structure of R varies.– R H
H3N +
The amino acids obtained by hydrolysis of proteins differ in respect to R (the side chain).
The properties of the amino acid vary as the structure of R varies.
Dr. Wolf's CHM 424

8. The major differences among the side chains concern: – R H
H3N +
The major differences among the side chains concern:
Size and shape Electronic characteristics
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9. General categories of a-amino acids
nonpolar side chains polar but nonionized side chains acidic side chains basic side chains
Dr. Wolf's CHM 424

10. General categories of a-amino acids
nonpolar side chains polar but nonionized side chains acidic side chains basic side chains
Dr. Wolf's CHM 424

11. Amino Acid R-groups Non-Polar Hydrophobic Polar Charged Uncharged
Tryptophan
Phenylalanine
Isoleucine
Tyrosine
Leucine
Valine
Methionine
Polar
Charged
Arginine (+)
Glutamic acid (-)
Aspartic Acid (-)
Lysine (+)
Histidine (+)
Uncharged
Cysteine
Proline
Serine
Glutamine
Asparagine
Ambivalent
Glycine
Threonine
Alanine
Dr. Wolf's CHM 424

12. - - Charged Amino Acids + + + Dr. Wolf's CHM 424 Arginine [Arg]
Glutamate [Glu]
Aspartate [Asp]
Lysine [Lys] +
Histidine [His]
Dr. Wolf's CHM 424

13. Hydrophobic Indexes Arginine Arg [R] -11.2 Glycine Gly [G] 0
Glutamic Acid Glu [E] -9.9
Aspartic Acid Asp [D] -7.4
Lysine Lys [K] -4.2
Histidine His [H] -3.3
Cysteine Cys [C] -2.8
Proline Pro [P] -0.5
Serine Ser [S] -0.3
Glutamine Gln [Q] -0.3
Asparagine Asn [N] -0.2
Glycine Gly [G] 0
Threonine Thr [T] 0.4
Alanine Ala [A] 0.5
Methionine Met [M] 1.3
Valine Val [V] 1.5
Leucine Leu [L] 1.8
Tyrosine Tyr [Y] 2.3
Isoleucine Ile [I] 2.5
Phenylalanine Phe [F] 2.5
Tryptophan Trp [W] 3.4
Dr. Wolf's CHM 424

14. Isoleucine Leucine Valine Tryptophan Methionine Lysine
Essential amino acids
Definition - Those amino acids that cannot be synthesized in the body in sufficient quantities for anabolic needs.
In humans,
Isoleucine Leucine Valine
Tryptophan Methionine Lysine
Phenylalanine Threonine Histidine
Dr. Wolf's CHM 424

15. C O – H H3N + Glycine (Gly or G)
Glycine is the simplest amino acid. It is the only one in the table that is achiral.
In all of the other amino acids in the table the  carbon is a chirality center.
Dr. Wolf's CHM 424

16. H O + – H3N C C O CH3 Alanine (Ala or A)
Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar and hydrophobic.
Dr. Wolf's CHM 424

17. H O + –
H3N C C O
CH(CH3)2
Valine
(Val or V)
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18. H O + –
H3N C C O
CH2CH(CH3)2
Leucine
(Leu or L)
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19. C O –
CH3CHCH2CH3 H
H3N +
Isoleucine
(Ile or I)
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20. H O + – H3N C C O CH3SCH2CH2 Methionine (Met or M)
The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat polarizable.
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21. H O + – H2N C Proline H2C CH2 (Pro or P) C H2
Proline is the only amino acid that contains a secondary amine function. Its side chain is nonpolar and cyclic.
Dr. Wolf's CHM 424

22. H O + – H3N C CH2 Phenylalanine (Phe or F)
The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group.
Dr. Wolf's CHM 424

23. H O + – H3N C Tryptophan CH2 (Trp or W)
The side chain in tryptophan (a nonpolar amino acid) is larger and more polarizable than the benzyl group of phenylalanine.
Dr. Wolf's CHM 424

24. General categories of a-amino acids
nonpolar side chains polar but nonionized side chains acidic side chains basic side chains
Dr. Wolf's CHM 424

25. The —CH2OH side chain in serine can be involved in hydrogen bonding.+ –
H3N C C O
CH2OH
Serine
(Ser or S)
The —CH2OH side chain in serine can be involved in hydrogen bonding.
Dr. Wolf's CHM 424

26. H O + – H3N C C O CH3CHOH Threonine (Thr or T)
The side chain in threonine can be involved in hydrogen bonding, but is somewhat more crowded than in serine.
Dr. Wolf's CHM 424

27. H O + – H3N C C O CH2SH Cysteine (Cys or C)
The side chains of two remote cysteines can be joined by forming a covalent S—S bond.
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28. H O + – H3N C Tyrosine CH2 (Tyr or Y)
The side chain of tyrosine is similar to that of phenylalanine but can participate in hydrogen bonding.
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29. H O + – H3N C Asparagine H2NCCH2 (Asn or N)
The side chains of asparagine and glutamine (next slide) terminate in amide functions that are polar and can engage in hydrogen bonding.
Dr. Wolf's CHM 424

30. C O – H
H3N +
H2NCCH2CH2
Glutamine
(Gln or Q)
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31. General categories of a-amino acids
nonpolar side chains polar but nonionized side chains acidic side chains basic side chains
Dr. Wolf's CHM 424

32. H O + – H3N C Aspartic Acid OCCH2 (Asp or D)
Aspartic acid and glutamic acid (next slide) exist as their conjugate bases at biological pH. They are negatively charged and can form ionic bonds with positively charged species.
Dr. Wolf's CHM 424

33. C O – H
H3N +
OCCH2CH2
Glutamic Acid
(Glu or E)
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34. General categories of a-amino acids
nonpolar side chains polar but nonionized side chains acidic side chains basic side chains
Dr. Wolf's CHM 424

35. H O + – H3N C C O Lysine + (Lys or K) CH2CH2CH2CH2NH3
Lysine and arginine (next slide) exist as their conjugate acids at biological pH. They are positively charged and can form ionic bonds with negatively charged species.
Dr. Wolf's CHM 424

36. H O + – Arginine H3N C C O (Arg or R) CH2CH2CH2NHCNH2 + NH2
Dr. Wolf's CHM 424

37. H O + – H3N C C O Histidine (His or H)
Histidine is a basic amino acid, but less basic than lysine and arginine. Histidine can interact with metal ions and can help move protons from one site to another.
CH2 NH N
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38. Stereochemistry of Amino Acids
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39. Configuration of -Amino Acids
Glycine is achiral. All of the other amino acids in proteins have the L-configuration at their carbon.
H3N + H R
CO2 –
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40. Amino Acids All DNA encoded aa are  All are chiral, except Glycine
R = H
All DNA encoded aa are usually L-
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41. Acid-Base Behavior of Amino Acids
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42. Recall
While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively.
How do we know this?
Dr. Wolf's CHM 424

43. For aa with basic R-groups, we require higher pHs, and
aa are high melting point solids! Why?
Answer = aa are ionic compounds under normal conditions
Isoelectric Point = concentration of zwitterion is at a maximum and the concentration of cations and anions is equal
For aa with basic R-groups, we require higher pHs, and
for aa with acidic R-groups, we require lower pHs
to reach the Isoelectric Point
Dr. Wolf's CHM 424

44. e.g. Isoelectric point for gly pH = 6.0 Asp pH = 3.0 Lys pH = 9.8
Isoelectric Point is the pH at which an aa or peptide carries no net charge.
i.e. [RCOO-] = [RNH3+]
So, for basic R-groups, we require higher pHs, and for acidic R-groups we require lower pHs
e.g. Isoelectric point for gly pH = 6.0
Asp pH = 3.0
Lys pH = 9.8
Arg pH = 10.8
Dr. Wolf's CHM 424

45. The properties of glycine:
high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent O OH
H2NCH2C ••
• • –
H3NCH2C +
more consistent with this
than this
Dr. Wolf's CHM 424

46. called a zwitterion or dipolar ion
Properties of Glycine
The properties of glycine:
high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent
more consistent with this
called a zwitterion or dipolar ion –
• • O
H3NCH2C •• +
Dr. Wolf's CHM 424

47. Acid-Base Properties of Glycine
The zwitterionic structure of glycine also follows from considering its acid-base properties.
A good way to think about this is to start with the structure of glycine in strongly acidic solution, say pH = 1.
At pH = 1, glycine exists in its protonated form (a monocation). O OH
H3NCH2C + ••
• •
Dr. Wolf's CHM 424

48. Acid-Base Properties of Glycine
Now ask yourself "As the pH is raised, which is the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is it the proton of the carboxyl group?"
You can choose between them by estimating their respective pKas.
typical ammonium ion: pKa ~9
typical carboxylic acid: pKa ~5 O OH
H3NCH2C + ••
• •
Dr. Wolf's CHM 424

49. Acid-Base Properties of Glycine
The more acidic proton belongs to the CO2H group. It is the first one removed as the pH is raised.
typical carboxylic acid: pKa ~5 O OH
H3NCH2C + ••
• •
Dr. Wolf's CHM 424

50. Acid-Base Properties of Glycine
Therefore, the more stable neutral form of glycine is the zwitterion. –
• • O
H3NCH2C •• +
typical carboxylic acid: pKa ~5 O OH
H3NCH2C + ••
• •
Dr. Wolf's CHM 424

51. Acid-Base Properties of Glycine
The measured pKa of glycine is 2.34.
Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an electron-withdrawing, acid-strengthening substituent on the  carbon.
typical carboxylic acid: pKa ~5 O OH
H3NCH2C + ••
• •
Dr. Wolf's CHM 424

52. Acid-Base Properties of Glycine
A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased further. –
• • O
H3NCH2C •• + –
• • O
H2NCH2C •• HO –
The pKa for removal of this proton is This value is about the same as that for NH4+ (9.3).
Dr. Wolf's CHM 424

53. Isoelectric Point pI O OH H3NCH2C +••
• •
The pH at which the concentration of the zwitterion is a maximum is called the isoelectric point. Its numerical value is the average of the two pKas.
The pI of glycine is 5.97.
pKa = 2.34 –
• • O
H3NCH2C •• +
pKa = 9.60 –
• • O
H2NCH2C ••
Dr. Wolf's CHM 424

54. Acid-Base Properties of Amino Acids
One way in which amino acids differ is in respect to their acid-base properties. This is the basis for certain experimental methods for separating and identifying them.
Just as important, the difference in acid-base properties among various side chains affects the properties of the proteins that contain them.
Table 27.2 gives pKa and pI values for amino acids with neutral side chains.
Dr. Wolf's CHM 424

55. Amino Acids with Neutral Side Chains– H
H3N +
pKa1 = pKa2 = 9.60 pI = 5.97
Glycine
Dr. Wolf's CHM 424

56. Amino Acids with Neutral Side Chains–
CH3 H +
pKa1 = pKa2 = 9.69 pI = 6.00
Alanine
Dr. Wolf's CHM 424

57. Amino Acids with Neutral Side Chains–
CH(CH3)2 H +
pKa1 = pKa2 = 9.62 pI = 5.96
Valine
Dr. Wolf's CHM 424

58. Amino Acids with Neutral Side Chains–
CH2CH(CH3)2 H +
pKa1 = pKa2 = 9.60 pI = 5.98
Leucine
Dr. Wolf's CHM 424

59. Amino Acids with Neutral Side Chains
pKa1 = pKa2 = 9.60 pI = 5.98 + –
Isoleucine
H3N C C O
CH3CHCH2CH3
Dr. Wolf's CHM 424

60. Amino Acids with Neutral Side Chains
pKa1 = pKa2 = 9.21 pI = 5.74 + –
Methionine
H3N C C O
CH3SCH2CH2
Dr. Wolf's CHM 424

61. Amino Acids with Neutral Side Chains– H +
CH2
H2C
C H2
pKa1 = pKa2 = pI = 6.30
Proline
Dr. Wolf's CHM 424

62. Amino Acids with Neutral Side Chains– H +
CH2
pKa1 = pKa2 = 9.13 pI = 5.48
Phenylalanine
Dr. Wolf's CHM 424

63. Amino Acids with Neutral Side Chains– H +
CH2 N
pKa1 = pKa2 = 9.39 pI = 5.89
Tryptophan
Dr. Wolf's CHM 424

64. Amino Acids with Neutral Side Chains
pKa1 = pKa2 = 8.80 pI = 5.41 + –
Asparagine
H3N C C O
H2NCCH2 O
Dr. Wolf's CHM 424

65. Amino Acids with Neutral Side Chains– H +
H2NCCH2CH2
pKa1 = pKa2 = 9.13 pI = 5.65
Glutamine
Dr. Wolf's CHM 424

66. Amino Acids with Neutral Side Chains–
CH2OH H +
pKa1 = pKa2 = 9.15 pI = 5.68
Serine
Dr. Wolf's CHM 424

67. Amino Acids with Neutral Side Chains
pKa1 = pKa2 = 9.10 pI = 5.60 + –
Threonine
H3N C C O
CH3CHOH
Dr. Wolf's CHM 424

68. Amino Acids with Neutral Side Chains– H +
CH2 OH
pKa1 = pKa2 = 9.11 pI = 5.66
Tyrosine
Dr. Wolf's CHM 424

69. Amino Acids with Neutral Side Chains–
CH2SH H +
pKa1 = pKa2 = 8.18 pI = 5.07
Cysteine
Dr. Wolf's CHM 424

70. Amino Acids with Ionizable Side Chains
pKa1 = pKa2 = 3.65 pKa3 = pI = 2.77 + –
Aspartic acid
H3N C C O
OCCH2 O –
For amino acids with acidic side chains, pI is the average of pKa1 and pKa2.
Dr. Wolf's CHM 424

71. Amino Acids with Ionizable Side Chains
pKa1 = pKa2 = 4.25 pKa3 = pI = 3.22 + –
Glutamic acid
H3N C C O
OCCH2CH2 – O
Dr. Wolf's CHM 424

72. Amino Acids with Ionizable Side Chains
H3N C O – H +
CH2CH2CH2CH2NH3
pKa1 = pKa2 = 8.95 pKa3 = pI = 9.74
Lysine
For amino acids with basic side chains, pI is the average of pKa2 and pKa3.
Dr. Wolf's CHM 424

73. Amino Acids with Ionizable Side Chains
H3N C O – H +
CH2CH2CH2NHCNH2
NH2
pKa1 = pKa2 = 9.04 pKa3 = pI = 10.76
Arginine
Dr. Wolf's CHM 424

74. Amino Acids with Ionizable Side Chains
H3N C O – H +
CH2 NH N
pKa1 = pKa2 = 6.00 pKa3 = pI = 7.59
Histidine
Dr. Wolf's CHM 424

75. Synthesis of Amino Acids
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76. From -Halo Carboxylic Acids
CH3CHCOH Br O
CH3CHCO
NH3 O + –
(65-70%)
NH4Br
H2O +
2NH3
Dr. Wolf's CHM 424

77. Strecker Synthesis CH3CH O NH4Cl CH3CHC NH2 N NaCN CH3CHCO NH3 O + –
(52-60%)
1. H2O, HCl, heat
2. HO–
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78. Using Diethyl Acetamidomalonate
OCH2CH3 H O
CH3CH2O
CH3CNH
Can be used in the same manner as diethyl malonate (Section 21.7).
Dr. Wolf's CHM 424

79. Example O CH3CH2OCCCOCH2CH3 CH3CNH H O 1. NaOCH2CH3 2. C6H5CH2Cl O
(90%) O
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80. O O Example HOCCCOH CH2C6H5 H3N + –CO2 O HBr, H2O, heat HCCOH CH2C6H5
(65%)
–CO2
HBr, H2O, heat O
CH3CH2OCCCOCH2CH3
CH2C6H5
CH3CNH
Dr. Wolf's CHM 424

81. Reactions of Amino Acids
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82. Acylation of Amino Group
The amino nitrogen of an amino acid can be converted to an amide with the customary acylating agents. O
CH3COCCH3 O
H3NCH2CO – + +
CH3CNHCH2COH O
(89-92%)
Dr. Wolf's CHM 424

83. Esterification of Carboxyl Group
The carboxyl group of an amino acid can be converted to an ester. The following illustrates Fischer esterification of alanine. O
H3NCHCO – +
CH3 +
CH3CH2OH
HCl
(90-95%) O
H3NCHCOCH2CH3 +
CH3 – Cl
Dr. Wolf's CHM 424

84. Ninhydrin Test OH O O H3NCHCO – + R + O N – O RCH + CO2 + H2O +
Amino acids are detected by the formation of a purple color on treatment with ninhydrin. OH O O
H3NCHCO – + R + O N – O
RCH +
CO2 +
H2O +
Dr. Wolf's CHM 424

85. Some Biochemical Reactions of Amino Acids
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86. Biosynthesis of L-Glutamic Acid
HO2CCH2CH2CCO2H O +
NH3
enzymes and
reducing coenzymes
HO2CCH2CH2CHCO2 –
NH3 +
This reaction is the biochemical analog of reductive amination (Section 22.10).
Dr. Wolf's CHM 424

87. Transamination via L-Glutamic Acid
HO2CCH2CH2CHCO2 –
NH3 +
CH3CCO2H O
L-Glutamic acid acts as a source of the amine group in the biochemical conversion of -keto acids to other amino acids. In the example to be shown, pyruvic acid is converted to L-alanine.
Dr. Wolf's CHM 424

88. Transamination via L-Glutamic Acid
HO2CCH2CH2CHCO2 –
NH3 +
CH3CCO2H O
enzymes
HO2CCH2CH2CCO2H O
CH3CHCO2 –
NH3 + +
Dr. Wolf's CHM 424

89. Mechanism O – HO2CCH2CH2CHCO2 CH3CCO2H + NH3
The first step is imine formation between the amino group of L-glutamic acid and pyruvic acid.
Dr. Wolf's CHM 424

90. Mechanism O – HO2CCH2CH2CHCO2 CH3CCO2H + NH3 – HO2CCH2CH2CHCO2 N –
Dr. Wolf's CHM 424

91. Formation of the imine is followed by proton removal at one carbon and protonation of another carbon. H
CH3CCO2 –
HO2CCH2CH2CCO2 N
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92. – HO2CCH2CH2CCO2 N – CH3CCO2 H H HO2CCH2CH2CCO2 N – CH3CCO2
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93. Hydrolysis of the imine function gives -keto glutarate and L-alanine.–
CH3CCO2 –
HO2CCH2CH2CCO2 N H
Hydrolysis of the imine function gives -keto glutarate and L-alanine.
Dr. Wolf's CHM 424

94. – HO2CCH2CH2CCO2 N – CH3CCO2 H H2O + NH3 – – HO2CCH2CH2CCO2 + CH3CCO2
Dr. Wolf's CHM 424

95. Biosynthesis of L-Tyrosine
L-Tyrosine is biosynthesized from L-phenylalanine.
A key step is epoxidation of the aromatic ring to
give an arene oxide intermediate.
CH2CHCO2 –
NH3 +
Dr. Wolf's CHM 424

96. Biosynthesis of L-Tyrosine
CH2CHCO2 –
NH3 + O
O2, enzyme
CH2CHCO2 –
NH3 +
Dr. Wolf's CHM 424

97. Biosynthesis of L-Tyrosine
CH2CHCO2 –
NH3 + O
enzyme
CH2CHCO2 –
NH3 + HO
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98. Biosynthesis of L-Tyrosine
Conversion to L-tyrosine is one of the major metabolic pathways of L-phenylalanine.
Individuals who lack the enzymes necessary to convert L-phenylalanine to L-tyrosine can suffer from PKU disease. In PKU disease, L-phenylalanine is diverted to a pathway leading to phenylpyruvic acid, which is toxic.
Newborns are routinely tested for PKU disease. Treatment consists of reducing their dietary intake of phenylalanine-rich proteins.
Dr. Wolf's CHM 424

99. Decarboxylation
Decarboxylation is a common reaction of -amino acids. An example is the conversion of L-histidine to histamine. Antihistamines act by blocking the action of histamine.
CH2CHCO2 –
NH3 +
N H N
Dr. Wolf's CHM 424

100. Decarboxylation N CH2CH2 NH2 N H –CO2, enzymes N – CH2CHCO2 N H + NH3
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101. Neurotransmitters – CO2OH
CO2 – H
H3N +
The chemistry of the brain and central nervous system is affected by neurotransmitters.
Several important neurotransmitters are biosynthesized from L-tyrosine.
L-Tyrosine
Dr. Wolf's CHM 424

102. Neurotransmitters – CO2
The common name of this compound is L-DOPA. It occurs naturally in the brain. It is widely prescribed to reduce the symptoms of Parkinsonism. +
H3N H H H HO OH
L-3,4-Dihydroxyphenylalanine
Dr. Wolf's CHM 424

103. Dopamine is formed by decarboxylation of L-DOPA. H2N H H H
Neurotransmitters H
Dopamine is formed by decarboxylation of L-DOPA.
H2N H H H HO OH
Dopamine
Dr. Wolf's CHM 424

104. Neurotransmitters H
H2N H H OH HO OH
Norepinephrine
Dr. Wolf's CHM 424

105. Neurotransmitters H
CH3NH H H OH HO OH
Epinephrine
Dr. Wolf's CHM 424

106. Peptides
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107. An amide bond of this type is often referred to as a peptide bond.
Peptides
Peptides are compounds in which an amide bond links the amino group of one -amino acid and the carboxyl group of another.
An amide bond of this type is often referred to as a peptide bond.
Dr. Wolf's CHM 424

108. Peptide bond formation
condensation
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109. Peptide bond formation
Primary Structure
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110. Alanine and Glycine
CH3 O C + H –
H3N O C H
H3N + –
Dr. Wolf's CHM 424

111. Alanylglycine CH3 O C H3N + H N –
Two -amino acids are joined by a peptide bond in alanylglycine. It is a dipeptide.
Dr. Wolf's CHM 424

112. Alanylglycine CH3 O C H3N + H N – N-terminus C-terminus Ala—Gly AG
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113. Alanylglycine and glycylalanine are constitutional isomers
CH3 O C
H3N + H N –
Alanylglycine
Ala—Gly AG H O C
H3N + N
CH3 –
Glycylalanine
Gly—Ala GA
Dr. Wolf's CHM 424

114. The peptide bond is characterized by a planar geometry.
Alanylglycine
CH3 O C
H3N + H N –
The peptide bond is characterized by a planar geometry.
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115. dipeptides, tripeptides, tetrapeptides, etc.
Higher Peptides
Peptides are classified according to the number of amino acids linked together.
dipeptides, tripeptides, tetrapeptides, etc.
Leucine enkephalin is an example of a pentapeptide.
Dr. Wolf's CHM 424

116. Tyr—Gly—Gly—Phe—Leu YGGFL
Leucine Enkephalin
Tyr—Gly—Gly—Phe—Leu YGGFL
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117. Oxytocin is a cyclic nonapeptide.
Ile—Gln—Asn
Tyr
Cys S
Cys—Pro—Leu—GlyNH2 1 2 3 4 5 6 7 8 9
C-terminus
N-terminus
Oxytocin is a cyclic nonapeptide.
Instead of having its amino acids linked in an extended chain, two cysteine residues are joined by an S—S bond.
Dr. Wolf's CHM 424

118. Oxytocin
S—S bond
An S—S bond between two cysteines is often referred to as a disulfide bridge.
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119. Introduction to Peptide Structure Determination
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120. Primary Structure
The primary structure is the amino acid sequence plus any disulfide links.
Dr. Wolf's CHM 424

121. Classical Strategy (Sanger)
1. Determine what amino acids are present and their molar ratios.
2. Cleave the peptide into smaller fragments, and determine the amino acid composition of these smaller fragments.
3. Identify the N-terminus and C-terminus in the parent peptide and in each fragment.
4. Organize the information so that the sequences of small fragments can be overlapped to reveal the full sequence.
Dr. Wolf's CHM 424

122. Amino Acid Analysis
Dr. Wolf's CHM 424 4

123. Amino acids are detected using ninhydrin.
Amino Acid Analysis
Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids.
The mixture is separated by ion-exchange chromatography, which depends on the differences in pI among the various amino acids.
Amino acids are detected using ninhydrin.
Automated method; requires only 10-5 to 10-7 g of peptide.
Dr. Wolf's CHM 424

124. Partial Hydrolysis of Proteins
Dr. Wolf's CHM 424 4

125. Partial Hydrolysis of Peptides and Proteins
Acid-hydrolysis of the peptide cleaves all of the peptide bonds.
Cleaving some, but not all, of the peptide bonds gives smaller fragments.
These smaller fragments are then separated and the amino acids present in each fragment determined.
Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis.
Dr. Wolf's CHM 424

126. Partial Hydrolysis of Peptides and Proteins
The enzymes that catalyze the hydrolysis of peptide bonds are called peptidases, proteases, or proteolytic enzymes.
Dr. Wolf's CHM 424

127. Trypsin
Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine.
lysine or arginine
NHCHC O R'
R" R
Dr. Wolf's CHM 424

128. phenylalanine, tyrosine, tryptophan NHCHC O
Chymotrypsin
Chymotrypsin is selective for cleaving the peptide bond to the carboxyl group of amino acids with an aromatic side chain.
phenylalanine, tyrosine, tryptophan
NHCHC O R'
R" R
Dr. Wolf's CHM 424

129. Carboxypeptidase
Carboxypeptidase is selective for cleaving the peptide bond to the C-terminal amino acid.
protein
H3NCHC O R +
NHCHCO – C
Dr. Wolf's CHM 424

130. End Group Analysis
Dr. Wolf's CHM 424 4

131. We need to know what the N-terminal and C-terminal amino acids are.
End Group Analysis
Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left.
We need to know what the N-terminal and C-terminal amino acids are.
The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis.
Several chemical methods have been developed for identifying the N-terminus. They depend on the fact that the amino N at the terminus is more nucleophilic than any of the amide nitrogens.
Dr. Wolf's CHM 424

132. Sanger's Method
The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene.
1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution (Section 23.5). F
O2N
NO2
Dr. Wolf's CHM 424

133. Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. F
O2N
NO2
NHCH2C
NHCHCO
CH3
NHCHC
CH2C6H5
H2NCHC O
CH(CH3)2 – +
O2N
NO2
NHCH2C
NHCHCO
CH3
NHCHC
CH2C6H5 O
CH(CH3)2 –
Dr. Wolf's CHM 424

134. Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group.
H3O+ O
O2N
NO2
NHCHCOH
CH(CH3)2
H3NCHCO–
CH3 +
H3NCH2CO– O
CH2C6H5
O2N
NO2
NHCH2C
NHCHCO
CH3
NHCHC
CH2C6H5 O
CH(CH3)2 –
Dr. Wolf's CHM 424

135. Insulin
Dr. Wolf's CHM 424 4

136. Insulin is a polypeptide with 51 amino acids.
It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids).
The following describes how the amino acid sequence of the B chain was determined.
Dr. Wolf's CHM 424

137. The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA
Dr. Wolf's CHM 424

138. The B Chain of Bovine Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Dr. Wolf's CHM 424

139. The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA
Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC
Dr. Wolf's CHM 424

140. The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
YTPKA
Dr. Wolf's CHM 424

141. The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA
Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC
Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence.
Dr. Wolf's CHM 424

142. The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
Dr. Wolf's CHM 424

143. The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALTLVCGERGFFYTPKA
Dr. Wolf's CHM 424

144. The sequence of the A chain was determined using the same strategy.
Insulin
The sequence of the A chain was determined using the same strategy.
Establishing the disulfide links between cysteine residues completed the primary structure.
Dr. Wolf's CHM 424

145. Primary Structure of Bovine Insulin
N terminus of A chain 5 15 10 20 25 30 S F V N Q H L C G A Y E R I K P T
C terminus of A chain
N terminus of B chain
C terminus of B chain
Dr. Wolf's CHM 424

146. The Edman Degradation and Automated Sequencing of Peptides
Dr. Wolf's CHM 424 4

147. 1. Method for determining N-terminal amino acid.
Edman Degradation
1. Method for determining N-terminal amino acid.
2. Can be done sequentially one residue at a time on the same sample. Usually one can determine the first 20 or so amino acids from the N-terminus by this method.
g of sample is sufficient.
4. Has been automated.
Dr. Wolf's CHM 424

148. The key reagent in the Edman degradation is phenyl isothiocyanate.
Dr. Wolf's CHM 424

149. Edman Degradation
Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid.
peptide
H3NCHC O R + NH
C6H5N C S +
Dr. Wolf's CHM 424

150. Edman Degradation peptide C6H5NHCNHCHC O R NH S peptide H3NCHC O R +
Dr. Wolf's CHM 424

151. The product is a phenylthiocarbamoyl (PTC) derivative.
Edman Degradation
peptide
C6H5NHCNHCHC O R NH S
The product is a phenylthiocarbamoyl (PTC) derivative.
The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the peptide.
Dr. Wolf's CHM 424

152. Edman Degradation peptide C6H5NHCNHCHC O R NH S HCl C6H5NH C S N CH R+ +
Dr. Wolf's CHM 424

153. The product is a thiazolone. Under the
Edman Degradation
The product is a thiazolone. Under the
conditions of its formation, the thiazolone rearranges to a phenylthiohydantoin (PTH)
derivative.
C6H5NH C S N CH R O
peptide
H3N + +
Dr. Wolf's CHM 424

154. Edman Degradation C N HN CH R O S C6H5
The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation.
C6H5NH C S N CH R O
peptide
H3N + +
Dr. Wolf's CHM 424

155. The Strategy of Peptide Synthesis
Dr. Wolf's CHM 424 4

156. General Considerations
Making peptide bonds between amino acids is not difficult.
The challenge is connecting amino acids in the correct sequence.
Random peptide bond formation in a mixture of phenylalanine and glycine, for example, will give four dipeptides.
Phe—Phe Gly—Gly Phe—Gly Gly—Phe
Dr. Wolf's CHM 424

157. N-Protected phenylalanine
General Strategy
1. Limit the number of possibilities by "protecting" the nitrogen of one amino acid and the carboxyl group of the other.
N-Protected phenylalanine
C-Protected glycine
NHCHCOH
CH2C6H5 O X
H2NCH2C Y
Dr. Wolf's CHM 424

158. 2. Couple the two protected amino acids.
General Strategy
2. Couple the two protected amino acids.
NHCH2C O Y
NHCHC
CH2C6H5 X
NHCHCOH
CH2C6H5 O X
H2NCH2C O Y
Dr. Wolf's CHM 424

159. General Strategy
3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus.
NHCH2C O Y
NHCHC
CH2C6H5 X
NHCH2CO O
H3NCHC
CH2C6H5 + –
Phe-Gly
Dr. Wolf's CHM 424

160. Amino Group Protection
Dr. Wolf's CHM 424 4

161. Protect Amino Groups as Amides
Amino groups are normally protected by converting them to amides.
Benzyloxycarbonyl (C6H5CH2O—) is a common protecting group. It is abbreviated as Z.
Z-protection is carried out by treating an amino acid with benzyloxycarbonyl chloride.
Dr. Wolf's CHM 424

162. Protect Amino Groups as Amides
CH2OCCl O
H3NCHCO
CH2C6H5 O – + +
1. NaOH, H2O
2. H+
NHCHCOH
CH2C6H5 O
CH2OC
(82-87%)
Dr. Wolf's CHM 424

163. Protect Amino Groups as Amides
NHCHCOH
CH2C6H5 O
CH2OC
is abbreviated as:
ZNHCHCOH
CH2C6H5 O
or Z-Phe
Dr. Wolf's CHM 424

164. Removing Z-Protection
An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by:
a) hydrogenolysis
b) cleavage with HBr in acetic acid
Dr. Wolf's CHM 424

165. Hydrogenolysis of Z-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH2OC
H2, Pd
H2NCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH3
CO2
(100%)
Dr. Wolf's CHM 424

166. HBr Cleavage of Z-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH2OC
HBr
H3NCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH2Br
CO2
(82%) + Br –
Dr. Wolf's CHM 424

167. The tert-Butoxycarbonyl Protecting Group
NHCHCOH
CH2C6H5 O
(CH3)3COC
is abbreviated as:
BocNHCHCOH
CH2C6H5 O
or Boc-Phe
Dr. Wolf's CHM 424

168. HBr Cleavage of Boc-Protecting Group
(CH3)3COC
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
H3NCHCNHCH2CO2CH2CH3
CH2C6H5 O
CO2
(86%) + Br –
CH2 C
H3C
Dr. Wolf's CHM 424

169. Carboxyl Group Protection
Dr. Wolf's CHM 424 4

170. Protect Carboxyl Groups as Esters
Carboxyl groups are normally protected as esters.
Deprotection of methyl and ethyl esters is by hydrolysis in base.
Benzyl esters can be cleaved by hydrogenolysis.
Dr. Wolf's CHM 424

171. Hydrogenolysis of Benzyl Esters
NHCHCNHCH2COCH2C6H5
CH2C6H5 O
C6H5CH2OC
H2, Pd
H3NCHCNHCH2CO
CH2C6H5 O
C6H5CH3
CO2
(87%) + –
CH3C6H5
Dr. Wolf's CHM 424

172. Peptide Bond Formation
Dr. Wolf's CHM 424 4

173. aa are covalently linked by amide bonds (Peptide Bonds)
The resulting molecules are called
Peptides & Proteins
Features of a Peptide Bond;
Usually inert
Planar to allow delocalisation
Restricted Rotation about the amide bond
Rotation of Groups (R and R’) attached to the amide bond is relatively free
Dr. Wolf's CHM 424

174. Primary Structure is the order (or sequence) of amino acid residues
aa that are part of a peptide or protein are referred to as residues.
Peptides are made up of about 50 residues, and do not possess a well-defined 3D-structure
Proteins are larger molecules that usually contain at least 50 residues, and sometimes The most important feature of proteins is that they possess well-defined 3D-structure.
Primary Structure is the order (or sequence) of amino acid residues
Peptides are always written and named with the amino terminus on the left and the carboxy terminus on the right
Dr. Wolf's CHM 424

175. Strong Acid Required to hydrolyse peptide bonds
Dr. Wolf's CHM 424

176. Cysteine residues create Disulfide Bridges between chains
This does not reveal Primary Structure
Dr. Wolf's CHM 424

177. The two major methods are:
Forming Peptide Bonds
The two major methods are:
1. coupling of suitably protected amino acids using N,N'-dicyclohexylcarbodiimide (DCCI)
2. via an active ester of the N-terminal amino acid.
Dr. Wolf's CHM 424

178. DCCI-Promoted Coupling
ZNHCHCOH
CH2C6H5 O
H2NCH2COCH2CH3 O +
DCCI, chloroform
ZNHCHC
CH2C6H5 O
NHCH2COCH2CH3
(83%)
Dr. Wolf's CHM 424

179. Mechanism of DCCI-Promoted Coupling
ZNHCHCOH
CH2C6H5 O +
C6H11N C
NC6H11
CH2C6H5 O
C6H11N C H
OCCHNHZ
Dr. Wolf's CHM 424

180. Mechanism of DCCI-Promoted Coupling
The species formed by addition of the Z-protected amino acid to DCCI is similar in structure to an acid anhydride and acts as an acylating agent.
Attack by the amine function of the carboxyl-protected amino acid on the carbonyl group leads to nucleophilic acyl substitution.
CH2C6H5 O
C6H11N C H
OCCHNHZ
Dr. Wolf's CHM 424

181. Mechanism of DCCI-Promoted Coupling
C6H11N C
C6H11NH H O +
ZNHCHC
CH2C6H5
NHCH2COCH2CH3
H2NCH2COCH2CH3 O
CH2C6H5 O
C6H11N C H
OCCHNHZ
Dr. Wolf's CHM 424

182. The Active Ester Method
A p-nitrophenyl ester is an example of an "active ester."
p-Nitrophenyl is a better leaving group than methyl or ethyl, and p-nitrophenyl esters are more reactive in nucleophilic acyl substitution.
Dr. Wolf's CHM 424

183. The Active Ester Method
ZNHCHCO
CH2C6H5 O
H2NCH2COCH2CH3 O
NO2 +
chloroform
ZNHCHC
CH2C6H5 O
NHCH2COCH2CH3
(78%) + HO
NO2
Dr. Wolf's CHM 424

184. Solid-Phase Peptide Synthesis: The Merrifield Method
Dr. Wolf's CHM 424 4

185. Solid-Phase Peptide Synthesis
In solid-phase synthesis, the starting material is bonded to an inert solid support.
Reactants are added in solution.
Reaction occurs at the interface between the solid and the solution. Because the starting material is bonded to the solid, any product from the starting material remains bonded as well.
Purification involves simply washing the byproducts from the solid support.
Dr. Wolf's CHM 424

186. The Solid Support
CH2 CH
The solid support is a copolymer of styrene and divinylbenzene. It is represented above as if it were polystyrene. Cross-linking with divinylbenzene simply provides a more rigid polymer.
Dr. Wolf's CHM 424

187. The Solid Support
CH2 CH
Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings.
Dr. Wolf's CHM 424

188. The Solid Support
CH2 CH
CH2Cl
The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2).
Dr. Wolf's CHM 424

189. The Solid Support
CH2 CH
CH2Cl
The chloromethylated resin is treated with the Boc-protected C-terminal amino acid. Nucleophilic substitution occurs, and the Boc-protected amino acid is bound to the resin as an ester.
Dr. Wolf's CHM 424

190. The Merrifield Procedure
CH2 CH
CH2Cl
BocNHCHCO R O –
Dr. Wolf's CHM 424

191. The Merrifield Procedure
CH2 CH
BocNHCHCO R O
Next, the Boc protecting group is removed with HCl.
Dr. Wolf's CHM 424

192. The Merrifield Procedure
H2NCHCO R
CH2 CH O
DCCI-promoted coupling adds the second amino acid
Dr. Wolf's CHM 424

193. The Merrifield Procedure
NHCHCO R O
CH2 CH
BocNHCHC R'
Remove the Boc protecting group.
Dr. Wolf's CHM 424

194. The Merrifield Procedure
CH2 CH
NHCHCO R O
H2NCHC R'
Add the next amino acid and repeat.
Dr. Wolf's CHM 424

195. The Merrifield Procedure
CH2 CH
NHCHCO R O
NHCHC R' C +
H3N
peptide
Remove the peptide from the resin with HBr in CF3CO2H
Dr. Wolf's CHM 424

196. The Merrifield Procedure
CH2 CH
CH2Br
NHCHCO R O
NHCHC R' C +
H3N
peptide –
Dr. Wolf's CHM 424

197. Merrifield automated his solid-phase method.
The Merrifield Method
Merrifield automated his solid-phase method.
Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in 68% yield.
Synthesized ribonuclease (124 amino acids) in reactions; 11,391 steps
Nobel Prize in chemistry: 1984
Dr. Wolf's CHM 424

198. Secondary Structures of Peptides and Proteins
Dr. Wolf's CHM 424 4

199. Levels of Protein Structure
Primary structure = the amino acid sequence plus disulfide links
Secondary structure = conformational relationship between nearest neighbor amino acids
 helix pleated  sheet
Dr. Wolf's CHM 424

200. Levels of Protein Structure
The -helix and pleated  sheet are both characterized by:
planar geometry of peptide bond
anti conformation of main chain
hydrogen bonds between N—H and O=C
Dr. Wolf's CHM 424

201. a-helixes
Intra-chain
H-bonds
Secondary Structure
Dr. Wolf's CHM 424

202. b-strands
Inter-chain
H-bonds
Secondary Structure
Dr. Wolf's CHM 424

203. Pleated  Sheet
Shown is a  sheet of protein chains composed of alternating glycine and alanine residues.
Adjacent chains are antiparallel.
Hydrogen bonds between chains.
van der Waals forces produce pleated effect.
Dr. Wolf's CHM 424

204. Pleated  Sheet
 Sheet is most commonly seen with amino acids having small side chains (glycine, alanine, serine).
80% of fibroin (main protein in silk) is repeating sequence of —Gly—Ser—Gly—Ala—Gly—Ala—.
 Sheet is flexible, but resists stretching.
Dr. Wolf's CHM 424

205.  Helix
Shown is an  helix of a protein in which all of the amino acids are L-alanine.
Helix is right-handed with 3.6 amino acids per turn.
Hydrogen bonds are within a single chain.
Protein of muscle (myosin) and wool (-keratin) contain large regions of -helix. Chain can be stretched.
Dr. Wolf's CHM 424

206. -helix -pleated sheets Secondary Structure
The Development of Regular patterns of Hydrogen Bonding, which result in distinct folding patterns
-helix
-pleated sheets
Dr. Wolf's CHM 424

207. Tertiary Structure of Peptides and Proteins
Dr. Wolf's CHM 424 4

208. Refers to overall shape (how the chain is folded)
Tertiary Structure
Refers to overall shape (how the chain is folded)
Fibrous proteins (hair, tendons, wool) have elongated shapes
Globular proteins are approximately spherical
most enzymes are globular proteins
an example is carboxypeptidase
Dr. Wolf's CHM 424

209. Tertiary Structure
This is the 3D structure resulting from further regular folding of the polypeptide chains using H-bonding, Van der Waals, disulfide bonds and electrostatic forces – Often detected by X-ray crystallographic methods
Globular Proteins – “Spherical Shape” , include Insulin, Hemoglobin, Enzymes, Antibodies
---polar hydrophilic groups are aimed outwards towards water, whereas non-polar “greasy” hydrophobic hydrocarbon portions cluster inside the molecule, so protecting them from the hostile aqueous environment Soluble Proteins
Fibrous Proteins – “Long thin fibres” , include Hair, wool, skin, nails – less folded e.g. keratin - the -helix strands are wound into a “superhelix”. The superhelix makes one complete turn for each 35 turns of the -helix.
Dr. Wolf's CHM 424

210. Bays or pockets in proteins are called Active Sites
In globular proteins this tertiary structure or macromolecular shape determines biological properties
Bays or pockets in proteins are called Active Sites
Enzymes are Stereospecific and possess Geometric Specificity
The range of compounds that an enzyme excepts varies from a particular functional group to a specific compound
Emil Fischer formulated the lock-and-key mechanism for enzymes
All reactions which occur in living cells are mediated by enzymes and are catalysed by
Some enzymes may require the presence of a Cofactor.
This may be a metal atom, which is essential for its redox activity.
Others may require the presence of an organic molecule, such as NAD+, called a Coenzyme.
If the Cofactor is permanently bound to the enzyme, it is called a Prosthetic Group.
Dr. Wolf's CHM 424

211. For a protein composed of a single polypeptide molecule, tertiary structure is the highest level of structure that is attained
Myoglobin and hemoglobin were the first proteins to be successfully subjected to completely successful X-rays analysis by J. C. Kendrew and Max Perutz (Nobel Prize for Chemistry 1962)
Quaternary Structure
When multiple sub-units are held together in aggregates by Van der Waals and electrostatic forces (not covalent bonds)
Hemoglobin is tetrameric myglobin
For example, Hemoglobin has four heme units, the protein globin surrounds the heme – Takes the shape of a giant tetrahedron – Two identical  and  globins.
The  and  chains are very similar but distinguishable in both primary structure and folding
Dr. Wolf's CHM 424

212. Hb monomer (or myoglobin)
Tertiary structure
Quaternary structure
Hb monomer (or myoglobin)
Hb a2b2 tetramer
Dr. Wolf's CHM 424

213. It is a metalloenzyme containing Zn2+ at its active site.
Carboxypeptidase
Carboxypeptidase is an enzyme that catalyzes the hydrolysis of proteins at their C-terminus.
It is a metalloenzyme containing Zn2+ at its active site.
An amino acid with a positively charged side chain (Arg-145) is near the active site.
Dr. Wolf's CHM 424

214. Carboxypeptidase tube model ribbon model Disulfide bond Zn2+ Arg-145
N-terminus
C-terminus
tube model
ribbon model
Dr. Wolf's CHM 424

215. What happens at the active site?•• O O
H2N
• • +
H3N
peptide C
NHCHC – + C
Arg-145 R O
H2N
Dr. Wolf's CHM 424

216. What happens at the active site?•• O O
H2N
• • +
H3N
peptide C
NHCHC – + C
Arg-145 R O
H2N
The peptide or protein is bound at the active site by electrostatic attraction between its negatively charged carboxylate ion and arginine-145.
Dr. Wolf's CHM 424

217. What happens at the active site?
Zn2+ •• O O
H2N
• • +
H3N
peptide C
NHCHC – + C
Arg-145 R O
H2N
Zn2+ acts as a Lewis acid toward the carbonyl oxygen, increasing the positive character of the carbonyl carbon.
Dr. Wolf's CHM 424

218. What happens at the active site?
Zn2+ •• O O
H2N
• • +
H3N
peptide C
NHCHC – + C
Arg-145 R O
H2N O
• • H
Water attacks the carbonyl carbon. Nucleophilic acyl substitution occurs.
Dr. Wolf's CHM 424

219. What happens at the active site?
Zn2+
H2N + C
Arg-145
H3N
peptide O + C ••
• • –
H3NCHC R O –
H2N +
Dr. Wolf's CHM 424

220. Coenzymes
Dr. Wolf's CHM 424 4

221. acid-base (transfer and accept protons) nucleophilic acyl substitution
Coenzymes
The range of chemical reactions that amino acid side chains can participate in is relatively limited.
acid-base (transfer and accept protons) nucleophilic acyl substitution
Many other biological processes, such as oxidation-reduction, require coenzymes, cofactors, or prosthetic groups in order to occur.
Dr. Wolf's CHM 424

222. NADH, coenzyme A and coenzyme B12 are examples of coenzymes.
Heme is another example.
Dr. Wolf's CHM 424

223. Heme Molecule surrounding iron is a type of porphyrin. N Fe H3C CH3
CH2CH2CO2H CH
CH2
H2C
HO2CCH2CH2
Molecule surrounding iron is a type of porphyrin.
Dr. Wolf's CHM 424

224. Myoglobin
Heme
N-terminus
C-terminus
Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen transport).
Dr. Wolf's CHM 424

225. Protein Quaternary Structure: Hemoglobin
Dr. Wolf's CHM 424 4

226. Protein Quaternary Structure
Some proteins are assemblies of two or more chains. The way in which these chains are organized is called the quaternary structure.
Hemoglobin, for example, consists of 4 subunits.
There are 2  chains (identical) and 2  chains (also identical).
Each subunit contains one heme and each protein is about the size of myoglobin.
Dr. Wolf's CHM 424

227. Protein Structure Primary structure is the amino acid sequence.
Secondary structure is how the amino acids in sequence fold up locally. Examples are a-helixes and b-strands and loops.
Tertiary structure is the 3-dimensional folding of the secondary structural elements and connecting loops in space.
Quaternary structure is the association of multiple subunits, each with a tertiary structure and each a unique gene product.
Dr. Wolf's CHM 424

228. Stabilization of Protein Structure
Electrostatic interactions involve the interaction of (+) and (-) charged side groups.
Hydrogen bonds involve sharing of a hydrogen atom between two eletronegative atoms (e.g., O, N).
Van der Waal’s forces are weak forces based on optimal overlap of adjacent electronic orbitals. Can be repulsive.
Hydrophobic interactions are, by far, the most powerful force stabilizing protein structure. Basis of force is entropy gain realized by burying hydrophobic residues.
Dr. Wolf's CHM 424

229. Cofactors
Cofactors are exogenous molecules that associate with proteins to yield full activity. In the absence of cofactor, protein is an apoprotein.
Co-enzymes are soluble and associate transiently with enzyme during catalytic cycle. An example is vitamin K in activation of blood clotting enzymes.
Prosthetic groups are covalently attached to the protein. Examples are heme, in hemoglobin, and riboflavin, in flavoproteins.
Dr. Wolf's CHM 424

230. Prof. Linus Pauling Dr. Frederick Sanger, Nobel Prize for Chemistry
                               
                               
Prof. Linus Pauling
Dr. Frederick Sanger,
Nobel Prize for Chemistry
1958 and 1980
Peptide sequencing
Prof. R. B. Merrifield
Nobel Prize for Chemistry 1984
Automated Peptide Synthesis
Dr. Wolf's CHM 424

231. Globular proteins fold so as to "bury" the hydrophobic side chains, minimizing their contact with water
Most polar residues face the outside of the protein and interact with solvent
Most hydrophobic residues face the interior of the protein and interact with each other
Packing of residues is close, but protein interiors contain some empty space
The empty space is in the form of small cavities
Globular Proteins
Dr. Wolf's CHM 424

232. Globular proteins fold so as to "bury" the hydrophobic side chains, minimizing their contact with water
Most polar residues face the outside of the protein and interact with solvent
Most hydrophobic residues face the interior of the protein and interact with each other
Packing of residues is close, but protein interiors contain some empty space
The empty space is in the form of small cavities
Globular Proteins
Dr. Wolf's CHM 424

233. Objectives
Diagram and describe the effect of oxygen on the position of iron relative to the heme plane.
Describe how cooperative binding of oxygen by hemoglobin improves its effectiveness as an oxygen carrier.
Describe the relationship between Hb structure to the Bohr effect and explain its physiological significance..
Discuss how carbon dioxide affects the affinity of Hb for oxygen and why this is physiologically significant.
Explain the effect of bisphosphoglycerate (BPG) on the affinity of Hb for oxygen and how this is related to altitude and HbF.
Explain how carbon monoxide (CO) binds to Hb and its affinity relative to that of oxygen..
Describe the molecular basis of thalassemias and the aberrant Hb that are produced in these diseases..
List three embryonic forms of Hb..
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234. Myoglobin: 2o and 3o aspects
Myoglobin is a single peptide chain of 153 residues arranged in eight a-helical regions labeled A-H.
The heme cofactor is the oxygen binding site so it is necessary for myoglobin’s function, oxygen storage in mammalian muscle tissue.
His E7 and F8 are important for binding the heme group within the protein and for stabilizing bound oxygen.
Dr. Wolf's CHM 424

235. Myoglobin and Hemoglobin
Mb is monomer, Hb is a tetramer (a2b2).
Hb subunits are structurally similar to Mb, with 8 a-helical regions, no b-strands and no water.
Both contain heme prosthetic group
Both Mb and Hb contain proximal and distal histidines.
Affinity of Mb for oxygen is high, affinity of Hb for oxygen is low.
Dr. Wolf's CHM 424

236. Myoglobin &Hemoglobin
Two related protein for O2 transportation.
Mb has one chain
Hb has four chains
Each chain has two parts: a globin ( protein) and a heme ( non-protein)
Dr. Wolf's CHM 424

237. Myoglobin An O2 transport protein in muscle
A Globin( globular soluble protein), 151 residues that contains 8 a-helices (A,B,C,…..H)
Contains a heme
prosthetic group
Binds heme in hydrophobic pocket.
Polar groups exposed to solvent, Non-polar groups buried.
Dr. Wolf's CHM 424

238. Myoglobin: 2o and 3o structure
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239. The Heme Prosthetic Group
• Protoporphyrin with Fe(II)
• Covalent attachment of Fe via His F8 side chain
• Additional stabilization via hydrophobic interaction
• Fe(II) state is active, Fe(III) [oxidized]
• Fe(II) atom in heme binds O2
Dr. Wolf's CHM 424

240. Binding of O2 to Heme
Binding of O2 to a free heme group is irreversible ( heme- heme sandwich)
Enclosure in a protein( globin) allows reversible binding
O2 has only limited solubility (1 X 10-4 M) in water
Solubility problem overcome by binding to proteins
• Binding of O2 alters heme structure
Bright scarlet color of blood in arteries
Dark purple color of blood in veins
Dr. Wolf's CHM 424

241. O O C C H C H C H C H C O O H C C H N N Fe(II) H C N N C H C H C H C H
The Heme Group - - O O C C H C H C H C H C O O 2 2 2 2 H C C H 3 3 N N
Fe(II)
Pyrrole ring H C N 2 N C H C H 3 C H C H C H 3 2
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242. N of His F8 binds to 5th coordination site on heme iron
Oxygen binds to 6th coordination site on heme iron
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243. His E7 acts as a gate to favor oxygen binding over carbon monoxide.
Dr. Wolf's CHM 424

244. Hemoglobin A tetrameric protein two a-chains (141 AA)
two b-chains (146 AA)
four heme cofactors, one in each chain
The a and b chains are homologous to myoglobin.
Oxygen binds to heme in hemoglobin with same structure as in Mb but cooperatively: as one O2 is bound, it becomes easier for the next to bind.
Dr. Wolf's CHM 424

245. Hemoglobin Ubiquitous O2 transport protein
A globular soluble protein, 2X2 chains (164 kDa)
a and b chains 44% identical
All helical secondary structure (like myoglobin)
abab quaternary structure
a-subunit 141 residues
b-subunit 146 residues
Extensive contacts between subunits
Mix of hydrophobic, H-bond, and ionic interactions
a1b1 (a2b2)- 35 residues, a1b2 (a2b1)- 19 residues
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246. Dr. Wolf's CHM 424

247. Each chain is in ribbon form.
The heme groups are in space filling form
Dr. Wolf's CHM 424

248. The myoglobin curve is hyperbolic. The hemoglobin curve is sigmoidal.
Oxygen Binding Curves
Hemoglobin and myoglobin respond differently to increase in O2 concentration.
Myoglobin shows normal saturation behavior while hemoglobin shows cooperative behavior. Each oxygen added to a heme of Hb makes addition of the next one easier.
The myoglobin curve is hyperbolic.
The hemoglobin curve is sigmoidal.
Dr. Wolf's CHM 424

249. Hemoglobin O2 Binding Curve
Binding curve is sigmoidal
Artery: high pO2, loading of protein
Vein: lower pO2, unloading from protein
P50(hemoglobin) = 26 torr, adjusts as needed!!
*Drastic change in pO2 over physiological range*
Dr. Wolf's CHM 424

250. Oxygen Binding Curves-2
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251. Hemoglobin Equilibriuma b b O2 a
H+,CO2,BPG R
(high affinity) T
(low affinity)
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252. A Quaternary Structure Change
One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding
This large change is caused by movement of Fe by only nm when oxygen binds
Dr. Wolf's CHM 424

253. Oxygen binding by hemoglobin
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254. Allosteric Effectors
The R or T state can be stabilized by the binding of ligands other than O2.
H+. Lower pH favors the T state which causes Hb to release bound O2. This is known as the Bohr Effect.
CO2. Release of CO2 lowers pH via conversion to HCO3-: CO2 + H2O  HCO3- + H+. Reinforces Bohr Effect
Bisphosphoglycerate (BPG). Regulation of activity via binding more strongly to T state, helps to release O2.
Increase in levels of BPG helps adaptation to high altitude- faster than making more hemoglobin. Also important in hypoxia diseases (e.g. anemia)
Dr. Wolf's CHM 424

255. Competition between oxygen and H+
The Bohr Effect
Competition between oxygen and H+
Discovered by Christian Bohr
Binding of protons diminishes oxygen binding
Binding of oxygen diminishes proton binding
Important physiological significance-O2 saturation of Hb responds to pH
Dr. Wolf's CHM 424

256. The Bohr Effect
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257. Carbon dioxide diminishes oxygen binding
Bohr Effect II
Carbon dioxide diminishes oxygen binding
CO2 produced in metabolically active tissue (requires oxygen)
Hydration of CO2 in tissues and extremities leads to proton production
CO2 + H2O  HCO H+
These protons are taken up by Hb forcing more oxygen to dissociate
The reverse occurs in the lungs
Dr. Wolf's CHM 424

258. Carbon Monoxide Poisoning
Heme Fe(II) binds many other small molecules with structures similar to O2 including: CO, NO, H2S
O2 is actually binds to these other molecules, particularly CO.
• When exposed to CO, even at low concentrations, O2 transport proteins will be filled with CO  limiting their vital O2 capacity.
Dr. Wolf's CHM 424

259. 2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin
The sigmoid binding curve is only observed in the presence of 2,3-BPG
Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector
Dr. Wolf's CHM 424

260. 2,3-bisphosphoglycerate (2,3-BPG) is a negative allosteric effector of O2 binding to Hb - binds tighter to deoxyHb
2,3-BPG
Dr. Wolf's CHM 424

261. Side view of Hb tetramer
Heme in hemoglobin a b b a
Heme prosthetic group
Side view of Hb tetramer
Dr. Wolf's CHM 424

262. Binding of oxygen to heme iron
Ferrous is reduced and +2 charge
Ferric is oxidized and +3 charge
Dr. Wolf's CHM 424

263. Effect of oxygen on heme iron
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264. Effect of oxygen on heme iron
Dr. Wolf's CHM 424

265. Effect of oxygen on heme iron
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266. Cooperativity
Oxygen binding to one subunit of Hb, increases the affinity of the other subunits for additional oxygens. In other words, the first one is the hardest, the rest are easy.
Example: square of postage stamps.
Book of four stamps. To pull first stamp, you have to break two edges.
To pull second stamp, you have to break only one edge.
To pull third stamp, you have to break only one edge.
To pull fourth stamp, you don’t have to break any edges.
Dr. Wolf's CHM 424

267. Sigmoid shape indicates positive cooperativityMb Hb
Sigmoid shape indicates positive cooperativity
Dr. Wolf's CHM 424

268. Bohr Effect O2 level in arterial blood O2 level in venous blood 7.4
7.0
O2 level in arterial blood
O2 level in venous blood
Dr. Wolf's CHM 424

269. Hb structural families
Alpha family -
a1, a2 - found in adult hemoglobins HbA1, HbA2.
z - found in embryonic hemoglobins Hb Gower 1 and Hb Portland.
Beta family -
b - found in adult hemoglobin HbA1.
d - found in adult hemoglobin HbA2.
g - found in fetal hemoglobin HbF.
e - found in embryonic hemoglobin Hb Gower 1 and Hb Gower 2
Dr. Wolf's CHM 424

270. CO2 effect
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271. Effect of BPG BPG is responsible for cooperativity.
High altitude increases BPG, pushing curve further to right
Dr. Wolf's CHM 424

272. a b a b b a Effect of BPG BPG Side view (T) Side view (R)
Dr. Wolf's CHM 424

273. Effect of BPG
His+
Lys+ -
BPG
His+
Lys+
Dr. Wolf's CHM 424

274. Hemoglobin Equilibriuma b b O2 a
H+,CO2,BPG R
(high affinity) T
(low affinity)
Dr. Wolf's CHM 424

275. Hemoglobins in normal adultsα α γ β α δ γ β α α δ α
HbA
HbF
HbA2
98%
~1%
<3.5%
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276. Dr. Wolf's CHM 424

277. Globin gene clusters
Dr. Wolf's CHM 424

278. Hb structural families
Alpha family -
a1, a2 - found in adult hemoglobins HbA1, HbA2.
z - found in embryonic hemoglobins Hb Gower 1 and Hb Portland.
q - (theta) newly discovered embryonic form.
Beta family -
b - found in adult hemoglobin HbA1.
d - found in adult hemoglobin HbA2.
g - found in fetal hemoglobin HbF.
e - found in embryonic hemoglobin Hb Gower 1 and Hb Gower 2
Dr. Wolf's CHM 424

279. FETAL AND NEONATAL ERYTHROPOIESIS
TABLE 1. Globin-chain development and composition
Developmental
stage
Hemoglobin
type
Globin-chain
composition
Embryo
Embryo to fetus
Fetus to adult
Adult
Gower 1
Gower 2
Portland
Fetal A A2
Zeta2 , epsilon2a
Alpha2, epsilon2
Zeta2, gamma2
Alpha2, gamma2
Alpha2, beta2
Alpha2, delta2
Alpha2, gamma2b
a This tetramer may be an epsilon tetrad.
b Fetal hemoglobin produced by adults has a different amino acid heterogeneity of the gamma chain at the 136 position than fetal hemoglobin
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280. Inherited Hemoglobin disorder
Definition: An inherited mutation of the globin genes leading to a qualitative or quantitative abnormality of globin synthesis
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281. The Thalassemias (quantitative)
Syndromes in which the rate of synthesis of a globin chain is reduced
beta thalassemia - reduced beta chain synthesis
alpha thalassemia – reduced alpha chain synthesis
Dr. Wolf's CHM 424

282. Alpha Thalassemias
Rare, since a gene is duplicated (four genes per diploid).
Usually more severe than beta thalassemia because
there is no substitute for a gene in adults.
Almost all a thalassemias are deletions
In a thalassemia major (a0a/a0a0) - occurrence of HbH (b4) and Hb Bart’s (g4).
BPG is ineffective in HbH & Hb Bart’s.
Dr. Wolf's CHM 424

283. Beta thalassemia Impaired production of beta chain
beta thalassemia minor – heterozygous (or trait)
beta thalassemia major - homozygous
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284. Beta thalassemia - heterozygous (minor or trait)
Target cell
Oval cell
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285. Beta thalassemia major
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286. More common, since b gene is present in only one copy per chromosome.
Beta Thalassemias
More common, since b gene is present in only one copy per chromosome.
Less severe than a thalassemia, since d chain can effectively substitute in adults.
The g chain can also persist into adulthood (HPFH).
In bd thal major (bd0/bd0) excess a chains do not form soluble homotetramers.
Dr. Wolf's CHM 424

287. Beta thalassemia major
No beta chain produced (no HbA)
Severe microcytic anemia occurs gradually in the first year of life
Marrow expansion
Iron overload
Growth failure and death
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288. Dr. Wolf's CHM 424

289. Alpha thalassemia / Normal /- Mild microcytosis /- - -/- -
Hemoglobin H disease
- -/- -
Hemoglobin Barts – Hydrops Fetalis
Dr. Wolf's CHM 424

290. Structural hemoglobinopathy (qualitative)
Amino acid substitution in the globin chain e.g. sickle hemoglobin (HbS)
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291. Sickle cell hemoglobin
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292. Dr. Wolf's CHM 424

293. Red Blood Cells from Sickle Cell Anemia
Deoxygenation of SS erythrocytes leads to intracellular hemoglobin polymerization, loss of deformability and changes in cell morphology.
OXY-STATE
DEOXY-STATE
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294. Sickle Cell Anemia – blood film
Sickle Cells
Erythroblasts
Howell-Jolly Body
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295. Fibres of Sickle Hemoglobin
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296. Fibres of Sickle Hemoglobin – cross section
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297. Hemoglobin S
Valine is exposed in deoxy-Hemoglobin
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298. Polymerization of HbS
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