Chapter 27 Amino Acids, Peptides, and Proteins

1
Chapter 27Amino Acids, Peptides, and Proteins
2
27.1Classification of Amino Acids
3
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.

4
Amino Acids
an ?-amino acid that is anintermediate in the
biosynthesisof ethylene
?
a ?-amino acid that is one ofthe structural
units present incoenzyme A
?
a ?-amino acid involved inthe transmission of
nerveimpulses
?
5
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.

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Table 27.1

• 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.

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Table 27.1

• The major differences among the side chains
  concern
• Size and shape Electronic characteristics

8
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

9
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

10
Table 27.1
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.

11
Table 27.1
O
H


O
H3N
C
C
CH3
Alanine
(Ala or A)

• Alanine, valine, leucine, and isoleucine have
  alkyl groups as side chains, which are nonpolar
  and hydrophobic.

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Table 27.1
O
H


O
H3N
C
C
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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Table 27.1
Phenylalanine
(Phe or F)

• The side chain in phenylalanine (a nonpolar amino
  acid) is a benzyl group.

14
Table 27.1

• The side chain in tryptophan (a nonpolar amino
  acid) is larger and more polarizable than the
  benzyl group of phenylalanine.

15
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

16
Table 27.1
O
H


O
H3N
C
C
CH2OH
Serine
(Ser or S)

• The CH2OH side chain in serine can be involved
  in hydrogen bonding.

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Table 27.1
O
H


O
H3N
C
C
CH2SH
Cysteine
(Cys or C)

• The side chains of two remote cysteines can be
  joined by forming a covalent SS bond.

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Table 27.1
Tyrosine
(Tyr or Y)

• The side chain of tyrosine is similar to that of
  phenylalanine but can participate in hydrogen
  bonding.

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Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

20
Table 27.1
Aspartic Acid
(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.

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Table 27.1
Glutamic Acid
(Glu or E)
22
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

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Table 27.1
O
H


O
H3N
C
C
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.

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Table 27.1
O
H


Arginine
O
H3N
C
C
(Arg or R)
CH2CH2CH2NHCNH2

NH2
25
Table 27.1
O
H


O
H3N
C
C
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.

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27.2Stereochemistry of Amino Acids
27
Configuration of ?-Amino Acids

• Glycine is achiral. All of the other amino acids
  in proteins have the L-configuration at their
  ??carbon.

28
27.3Acid-Base Behavior of Amino Acids
29
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?
30
Properties of Glycine

• The properties of glycine
• high melting point (when heated to 233C it
  decomposes before it melts)solubility soluble
  in water not soluble in nonpolar solvent

31
Properties of Glycine

• The properties of glycine
• high melting point (when heated to 233C it
  decomposes before it melts)solubility soluble
  in water not soluble in nonpolar solvent

more consistent with this
called a zwitterion or dipolar ion
32
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).

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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.

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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
35
Acid-Base Properties of Glycine

• Therefore, the more stable neutral form of
  glycine is the zwitterion.

typical carboxylic acid pKa 5
36
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
37
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.

• The pKa for removal of this proton is 9.60.This
  value is about the same as that for NH4 (9.3).

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Isoelectric Point pI

• 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
pKa 9.60
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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.

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Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.34pKa2 9.60pI 5.97
Glycine
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Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.34pKa2 9.69pI 6.00
Alanine
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Table 27.3 Amino Acids with Ionizable Side Chains
O
H
pKa1 1.88pKa2 3.65pKa3 9.60 pI 2.77


H3N
O
Aspartic acid
C
C

• For amino acids with acidic side chains, pI is
  the average of pKa1 and pKa2.

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Table 27.3 Amino Acids with Ionizable Side Chains
pKa1 2.18pKa2 8.95pKa3 10.53pI 9.74
Lysine

• For amino acids with basic side chains, pI is the
  average of pKa2 and pKa3.

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27.4Synthesis of Amino Acids
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From ?-Halo Carboxylic Acids
H2O

2NH3
46
Strecker Synthesis
NH4Cl
NaCN
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27.5Reactions of Amino Acids
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Acylation of Amino Group

• The amino nitrogen of an amino acid can be
  converted to an amide with the customary
  acylating agents.

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Esterification of Carboxyl Group

• The carboxyl group of an amino acid can be
  converted to an ester. The following illustrates
  Fischer esterification of alanine.

CH3CH2OH
HCl
50
Ninhydrin Test

• Amino acids are detected by the formation of a
  purple color on treatment with ninhydrin.

CO2
H2O
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27.6Some Biochemical Reactionsof Amino Acids
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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.
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Biosynthesis of L-Tyrosine
O2, enzyme
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Biosynthesis of L-Tyrosine
enzyme
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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.

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Neurotransmitters

• The chemistry of the brain and central nervous
  system is affected by neurotransmitters.
• Several important neurotransmitters are
  biosynthesized from L-tyrosine.

L-Tyrosine
57
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
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Neurotransmitters
H

• Dopamine is formed by decarboxylation of L-DOPA.

H2N
H
H
H
HO
OH
Dopamine
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27.7Peptides
60
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.

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Alanine and Glycine
62
Alanylglycine

• Two ?-amino acids are joined by a peptide bond in
  alanylglycine. It is a dipeptide.

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Alanylglycine
N-terminus
C-terminus
AlaGly
AG
64
Alanylglycine and glycylalanine are
constitutional isomers
Alanylglycine AlaGly AG
Glycylalanine GlyAla GA
65
Alanylglycine

• The peptide bond is characterized by a planar
  geometry.

66
Higher Peptides

• Peptides are classified according to the number
  of amino acids linked together.
• dipeptides, tripeptides, tetrapeptides, etc.
• Leucine enkephalin, an endorphin, is an example
  of a pentapeptide.

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Leucine Enkephalin
TyrGlyGlyPheLeuYGGFL
68
Oxytocin
C-terminus
N-terminus

• Oxytocin is a cyclic nonapeptide that stimulates
  uterine contractions.
• Instead of having its amino acids linked in an
  extended chain, two cysteine residues are joined
  by an SS bond.

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Oxytocin
SS bond
An SS bond between two cysteines isoften
referred to as a disulfide bridge.
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27.8Introduction to Peptide Structure
Determination
71
Primary Structure

• The primary structure is the amino acid sequence
  plus any disulfide links.

72
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.

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27.12Insulin
74
Insulin

• 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.

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The B Chain of Bovine Insulin

• Phenylalanine (F) is the N terminus.
• Pepsin-catalyzed hydrolysis gave the four
  peptides FVNQHLCGSHL VGAL VCGERGF YTPKA

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The B Chain of Bovine Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
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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 AL
  Y YLVC

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The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
YTPKA
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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 AL
  Y YLVC
• Trypsin-catalyzed hydrolysis gave GFFYTPK which
  completes the sequence.

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The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
GFFYTPK
YTPKA
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The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALYLVCGERGFFYTPKA
82
Insulin

• The sequence of the A chain was determined using
  the same strategy.
• Establishing the disulfide links between cysteine
  residues completed the primary structure.

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Primary Structure of Bovine Insulin
N terminus of A chain
C terminus of A chain
N terminus of B chain
C terminus of B chain
84
27.13The Edman Degradation and Automated
Sequencing of Peptides
85
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.
• 3. 10-10 g of sample is sufficient.
• 4. Has been automated.

86
Edman Degradation

• The key reagent in the Edman degradation is
  phenyl isothiocyanate.

87
Edman Degradation

• Phenyl isothiocyanate reacts with the amino
  nitrogen of the N-terminal amino acid.

88
Edman Degradation

89
Edman Degradation
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.

90
Edman Degradation
HCl

91
Edman Degradation
The product is a thiazolone. Under
the conditions of its formation, the
thiazolonerearranges to a phenylthiohydantoin
(PTH) derivative.

92
Edman Degradation

• The PTH derivative is isolated and identified.
  The remainder of the peptide is subjected to a
  second Edman degradation.

93
27.14The Strategy of Peptide Synthesis
94
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.
• PhePhe GlyGly PheGly GlyPhe

95
General Strategy

• 1. Limit the number of possibilities by
  "protecting" the nitrogen of one amino acid and
  the carboxyl group of the other.

96
General Strategy

• 2. Couple the two protected amino acids.

97
General Strategy

• 3. Deprotect the amino group at the N-terminus
  and the carboxyl group at the C-terminus.

Phe-Gly
98
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.

99
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

100
Hydrogenolysis of Z-Protecting Group
101
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.

102
Hydrogenolysis of Benzyl Esters
103
27.17Peptide Bond Formation
104
DCCI-Promoted Coupling

105
27.18Solid-Phase Peptide SynthesisThe
Merrifield Method
106
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.

107
The Solid Support

• 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.

108
The Solid Support

• Treating the polymeric support with chloromethyl
  methyl ether (ClCH2OCH3) and SnCl4 places ClCH2
  side chains on some of the benzene rings.

109
The Solid Support

• The side chain chloromethyl group is a benzylic
  halide, reactive toward nucleophilic substitution
  (SN2).

110
The Solid Support

• 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.

111
The Merrifield Procedure
112
The Merrifield Procedure

• Next, the Boc protecting group is removed with
  HCl.

113
The Merrifield Procedure

• DCCI-promoted coupling adds the second amino acid

114
The Merrifield Procedure

• Remove the Boc protecting group.

115
The Merrifield Procedure

• Add the next amino acid and repeat.

116
The Merrifield Procedure

• Remove the peptide from the resin with HBr in
  CF3CO2H

117
The Merrifield Procedure

118
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
  1969. 369 reactions 11,391 steps
• Nobel Prize in chemistry 1984

119
27.19Secondary Structuresof Peptides and
Proteins
120
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

121
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 NH and OC

122
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.

123
? 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.

124
27.20Tertiary Structureof Peptides and Proteins
125
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

126
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.

127
Carboxypeptidase
Disulfide bond
Zn2
Arg-145
N-terminus
C-terminus
tube model
ribbon model
128
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N
129
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N

• The peptide or protein is bound at the active
  site by electrostatic attraction between its
  negatively charged carboxylate ion and
  arginine-145.

130
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N

• Zn2 acts as a Lewis acid toward the carbonyl
  oxygen, increasing the positive character of the
  carbonyl carbon.

131
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N

• Water attacks the carbonyl carbon. Nucleophilic
  acyl substitution occurs.

132
What happens at the active site?
H2N

Arg-145
C
133
27.21Coenzymes
134
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.

135
Coenzymes

• NADH, coenzyme A and coenzyme B12 are examples of
  coenzymes.
• Heme is another example.

136
Heme

• Molecule surrounding iron is a type of porphyrin.

137
Myoglobin
Heme

• Heme is the coenzyme that binds oxygen in
  myoglobin (oxygen storage in muscles) and
  hemoglobin (oxygen transport).

138
27.22Protein Quaternary StructureHemoglobin
139
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.