David L. Nelson and Michael M.

Cox

LEHNINGER
PRINCIPLES OF BIOCHEMISTRY
th Edition

CHAPTER 3

Amino Acids, Peptides, and Proteins

1
2008 W. H. Freeman and Company

Structure, Assembly and Function of Amino Acids

I. Amino Acid
(building block monomers)

II. Peptide
III. Protein
(linking of amino acids) (linking of peptides)

I. Structure of Amino Acids

Functional Group Components:
Carboxylate C-terminus
Ammonium
N-terminal

chiral
C
side-chain
-20 naturally occurring L-amino acids
(side-chain single difference)
- amino acids are chiral molecules
capable of rotating plane polarized light
(optical activity measured by polarimetry)
- In there neutral state amino acids are
zwitterionic

R/S-Configuration?
3
Stereochemical Relationship?

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

7

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

I. Structure of Amino Acids

Nomenclature and Properties:

Post-Synthesis Modifications of Amino Acids

Enzymes catalyze amino acid modifications to alter structure and function in
biological processes
I. Acylation (O-Acylation-ester, N-acylation-amides and S-acylation thioesters)

II. Alkylation (Methylation- terminal amines, Prenylation-terpenes)

10

Post-Synthesis Modifications of Amino Acids

Enzymes catalyze amino acid modifications to alter structure and function in
biological processes
III. Redox Reactions (Oxidation, Reduction, Hydroxylation)

IV. Functional Group Transfer Reactions (Glycosylation, Phosphorylation,

Sulfonation, Iodination, Nitrosylation, Selenoylation)

11

Uncommon Amino Acids found in Nature

Natural Amino Acids Undergo post-synthesis
modifications for specific biological function
Collagen
(connective tissues)

Collagen
(connective tissues)
Myosin
(motor protein)

Prothrombin
(blood coagulation)

Elastin
(elastic protein)

Glutathione peroxidase
(anti-oxidant protein)
12

Synthetic Un-natural Amino Acids function as Molecular Probes to

Study Protein Structure & Function
For a review: Bioconjugate Chem. 2009, 20, 1281-1295.

Modified amino acids for

cycloaddition chemistry

Modified amino acids

as radical initiators
Modified amino acids with
photoreactive groups

Modified amino acids as

biophysical probes

Modified amino acids as

redox agents

13

Synthetic Un-natural Amino Acids function as Molecular Probes to

Study Protein Structure & Function
Examples:

Reverse Turn Mimics in Stabilizing

Ribonuclease A Structure
J. Am. Chem. Soc. 2002, 124, 8522

Structural Studies in
Prenylated GTPases
Science 2003, 302, 646.

Metal chelating amino acids

in metalloproteins
J. Am. Chem. Soc. 131, 2481.
14

Physical Properties of Amino Acids

-Side-chain aromatic chromophores absorb UV light
UV-VIS spectrophotometer
UV spectra

Beerslaw=logIo=cl
(Absorption,A)I

Use:
UV absorption of aromatic amino acid residues has applications in HPLC
purification, determining concentration of peptides or proteins and CD 15
spectroscopic structural determination of peptides and proteins

Physical Properties of Amino Acids

- Amino Acids are chiral with the ability to rotate plane polarized light
Optical Rotation Measurements and Amino Acid Enantiomeric Purity

polarimetry

Optical Purity

Specific rotation
obs

ee%=100*([R][S])
([R]+[S])
= Specific rotation of sample

Resolution of a Racemic Mixture of Amino

Acids

Specific rotation pure

enantiomer

16

Physical Properties of Amino Acids (contd)

Separation of Amino Acids by Chromatography
Thin-Layer Chromatography: the process of separating
amino acids on the basis of their polarity
Non-polar amino acids tend to migrate quicker on the polar silica
gel stationary phase
Polar amino acids tend to migrate slower on the polar silica gel
stationary phase
Polar organic solvents (MeOH, EtOH and acetone) elute a.a. best
The Ninhydrin Test can be used to detect a.a. on TLC

Physical Properties of Amino Acids (contd)

Separation of Amino Acids by Chromatography
Ion-Exchange Chromatography: the process of separating amino acids based on electric charge
Cation-exchange resins have a ve charged stationary phase and can be used to isolate +ve charged a.a.
Anion-exchange resins have a +ve charged stationary phase and can be used to isolate ve charged a.a.

Aminoacids

Amino
acids

Note:IonPairingbuffer
(triethylammoniumacetate,TEAA)
canbeusedtoeluteaminoacids
fromstationaryphase

Acid/Base Properties of Amino Acids

- Weak acid/base properties due to main & side-chain acidic/basic
functional groups
I. Ionic Structure

II. Amphoteric Structure

- acid/base properties

19

pKa Values for Amino Acids are based on main & side-chain
acidic/basic functional groups

20

Titration Curves to determine the pKa and pH of amino

acids in solution
Keq = Ka

HA

H+ + A-

Which pH will Gly be protonated and nonprotonated form?

Buffering region: region in which smallest
changes in pH occur with increasing [H+] or [OH-]
-mid-point of the titration curve= iso-electric
point, pI
pH = pKa at which a.a. are neutral (point at
which concentration of two species on either
side of equilibbrium is same so Conc of A- is
21
equalt to HA

Titration Curves Can be Useful in Predicting Overall Charge

of Amino Acids in Aqueous Solution
A Case Example: Ionization of Histidine

pKa = pH = pI = (pKa)n
n
7.59 = 6.0 + 9.17
2
1. Provide the ionic structure of His at the pI ?
2. Provide the ionic structure of His in a
mixture of 0.042 M NaH2PO4 and 0.058 M
Na2HPO4 phosphate buffer , pH = 7.4
22

Titration Curves Can be Useful in Predicting Overall Charge

of Amino Acids in Aqueous Solution
A Case Example: Ionization of Glutamic Acid

pKa = pH = pI = (pKa)n
n
3.22 = 2.19 + 4.25
2
1. Provide the ionic structure of Glu at the pI ?
2. Provide the ionic structure of Glu in a
mixture of 0.042 M NaH2PO4 and 0.058 M
Na2HPO4 phosphate buffer , pH = 7.4
23

Chemical Reactivity of Amino Acids

24

David L. Nelson and Michael M. Cox

LEHNINGER
PRINCIPLES OF BIOCHEMISTRY
Sixth Edition

CHAPTER 3

Amino Acids, Peptides, and Proteins

Optional Problems: 1-6

25

David L. Nelson and Michael M. Cox

LEHNINGER
PRINCIPLES OF BIOCHEMISTRY
Sixth Edition

CHAPTER 22

Biosynthesis of Amino Acids,

Nucleotides, and Related Molecules
26
2008 W. H. Freeman and Company

Biosynthesis of the 20 Naturally Occuring Amino Acids

I.

Glycolysis

(glucose metabolism and cellular energy in cytosol)

II.

Phosphate Pentose
Pathway

(amino acid/nucleic acid biosynthesis in cytosol)

III.

Citric Acid Cycle (Krebs Cycle)

(key metabolic pathway in amino acid
degradation for energy source in
mitochondrion-eukaryotic & cytosolprokaryotes)

27

Note:
-Half of the naturally occurring
amino acids in mammals are
biosynthesized (non-essential
AA)
- Essential amino acids are
obtained from diet
- Biosynthetic pathways for
amino acids are found in
bacteria

Identify the Metabolic pathway associated with

each amino acid biosynthetic family ?

- Amino Acids are synthesized

from a metabolic precursor
found in :
i.Glycolysis
ii.Citric Acid Cycle
iii.Pentose Phosphate
28
Pathway

Enzymes known to catalyze Amino Acid Metabolic Reactions

1. Dehydrogenases: Catalyzes the elimination of water
2. Synthases: Catalyzes the condensation reactions in making of C-C
3. Kinases: Catalyzes the transfer of P in the presence of ATP
4. Phosphatases/Phosphorylases: Catalyzes the hydrolysis of P in the presence of ATP
5. Reductases: Catalyzes reduction reactions in the presence of a reducing agent
6. Oxidases: Catalyzes oxidation reactions in the presence of an oxidizing agent
7. Dioxygenases: Catalyzes the removal of dioxygen from a molecule
8. Aminotransferases: Catalyze transamination reactions
9. Transferases: Catalyzes functional group transfer reactions
10. Lyases: Catalyzes the cleavage of covalent bonds in molecules
11. Ligases: Catalyzes the ligation (bond forming) reactions
12. Isomerases: Catalyzes the isomerization/rearrangement reactions
13. Mutases: Catalyzes the isomerization/rearrangement reactions
14. Cyclohydrolases: Catalyzes the ring opening hydrolysis reactions

29

Co-enzymes in Amino Acid Metabolic Reactions

co-enzymes are organic compounds that assist in the catalytic activity of
enzymes
Co-enzyme often
conjugated with
transaminase Co-enzyme involved
activity
in redox activity
Co-enzyme involved
in Leu metabolism

Co-enzyme involved
in methyl group
transfer

Co-enzyme involved
in functional group
transfer
30

Enzymes may contain additional elements or functional

groups which help catalyze reactions
Ion Cofactors
Metal Ion cofactors help
catalyze enzymatic activity
through covalent coordination
bonds

Reaction Classes in Metabolism

(1) Acid-Base Reactions
2 NaOH + H2SO4 2 H2O + Na2SO4

(4) Free-radical Reactions

hvor

(2) C-C Reactions

(5) Functional Group Transfer Reactions
atypicalReductiveAminationReaction

atypicalClaisenCondensationReaction

(6) Oxidation and Reduction Reactions

(3) Rearrangement Reactions

i.e. Claisen Rearrangement

(7) Pericyclic Reactions

32

 -Ketoglutarate as Starting Material in the biosynthesis of

Glutamate, Glutamine, Proline and Arginine in Bacteria
- Part of the citric acid cycle

33

Reductive amination and amminolysis of -ketoglutarate leads to

L-Gln via L-Glu intermediate

NADPH

NADP

Mechanism ?

Imine Intermediate
I. Amination
II. Reduction
Intermolecular Reductive Amination
- Identify the co-enzymes for the transformations??

34

Glu is precursor to Pro

synthesis
Phosphorylation
/activation

Reduction

Mechanism

Intramolecular Reductive Amination

35

Glu is precursor to Arg

synthesis
Mechanism

PG

Reductive
Amination
Amide
Hydrolysis

36

3-Phosphoglycerate as Starting Material in the

biosynthesis of Serine, Glycine and Cysteine in Bacteria
- as part of glycolysis

37

Biosynthesis of Ser and Gly from 3Phosphoglycerate

Ser Gly interconversion
provide a plausible mechanism ?

oxidation

Reductive
amination

NH4+
NADPH
NAD

NADHNAD, CO2, NH4+

Phosphate
hydrolysis

38

Ser Gly interconversion

mechanism

39

Biosynthesis of Cys from

Ser in Mammals

nucleophile ?
Thioether
formation

Cys + 1C

Redox reaction

40

Oxaloacetate and Pyruvate as starting materials in the

biosynthesis of Nine Amino Acids

41

Biosynthesis of Aspartate from Oxaloacetate

NH4+
NADPH NADP

Provide the Intermediate for this Transformation ?

42

Conversion of Asp to Lys

phosphorylation

reduction

Aldol
Condensation
reduction
43

Conversion of Asp to Lys

Mechanism for Aldol Condensation
OH
O

H+

OH

H2
CH C C

H2C

CO2-

NH2
Asp semialdehyde
O
H2C
H
B-

OH

H2
H2
CH C C C
H
NH2
Aldol product

CO2-

CO2pyruvate

44

Conversion of Asp to Lys

(contd)

Cyclization/
Dehydration

Reduction

Mechanism of formation ?
Hydrolysis/
Acylation

NH4+
NADPH NADP
Reductive
Amination

45

Conversion of Asp to Lys

(contd)

NH4+
NADPH NADP

Reductive Amination

Hydrolysis

Epimerization
Sterechemical
Relationship ?
Chirality ?

Decarboxylation
46

Conversion of Asp to Lys

Mechanism for dihydroxypicolinate synthesis
and Lys formation

47

Conversion of Asp to Met

Asp Asp--P

Mechanism ?

Reduction

Acylation

Cys Addn

48

Conversion of Asp to Met

(contd)
Mechanism ?

Reduction/elimination

Methylation

49

Conversion of Asp to Thr

Mechanism ?

Elimination/H
ydrolysis

Phosphorylation

Reduction Asp-CHO
Reduction
Asp-P
Phosphorylation
Asp

50

Conversion of pyruvate
and -ketobutyrate to Ile
and Val
Elimination/
Imine hydrolysis

Claisen
Condensation

Provide a Mechanism ?
51

Conversion of pyruvate
and -ketobutyrate to Ile
and Val
Mechanism for the Claisen Condensation of pyruvate to -acetolactate?

O
-

O 2C

H+
CH2

H
pyruvate

B-

O2C

OH
CH2

keto-isomer

O2C
enol-isomer

H3C

OH
CO2-

pyruvate

O2C
acetolactate

52

Conversion of pyruvate
and -ketobutyrate to Ile
and Val (contd)

reduction

reduction

53

Conversion of pyruvate
and -ketobutyrate to Ile
and Val (contd)

dehydration

NH4+
NADPHNAD
Reductive
Amination

54

Keto-isovalerate is an
intermediate for the
biosynthesis of Leu

rearrangement

Claisen Condensation/
Hydrolysis
Mechanism ?

oxidation

NH4+
NADPHNAD

Reductive
amination

55

Conversion of keto-isovalerate to a-isopropylmalate

Claisen Condensation/ Hydrolysis
Mechanism
O
CO2

CO2H

H2C

CoA
Claisen Condensation

O
keto-isovalerate

H2C
H

B-

O
OH
isopropylmalate-CoA

Hydrolysis
CoA
acetyl CoA

CoA

H2O

CO2H

OH

O
OH
isopropylmalate

56

Phosphoenolpyruvate and Erythrose 4-phosphate as

starting materials in the biosynthesis of Tryptophan,
Phenylalanine and Tyrosine
- As part of glycolysis

57

Phosphophenol
Conversion of
pyruvate
chorismate to Tyr,
+
Phe
Erythrose 4phosphate

Claisen type
Rearrangement
Mechanism ?

Decarboxylation/
aromatization
Mechanism ?

Reductive
Amination

58

Conversion of chorismate to Tyr, Phe

Mechanism for the Claisen Type Rearrangement followed by decarboxylation and aromatization

Claisen Rearrangement

Reductive amination

59

Phosphophenol
Conversion of
pyruvate
+
chorismate to Trp
Nucleophilic Substituion/ Erythrose 4phosphate
Claisen Rearrangement

NH3

Provide Mechanism ?
Nucleophilic
Displacement

Rearrangement
Mechanism ?
Provide Mechanism
for last 2 steps

Cyclization

Nucleophilic
Displacement
with Ser

60

Mechanism to
Synthesis of
Trp

61

Ribose 5-phosphate as starting materials in the

biosynthesis of Histidine
- As part of the phosphate pentose pathway

62

Conversion of
PRPP to Histidine
Nucleophilic Aromatic
Substitution Reaction

Phosphate Hydrolysis
63

Conversion of
PRPP to Histidine
Imine Hydrolysis

Reductive
Amination

Carbohydrate Rearrangement
(to be discussed with carbohydrates)
64

Conversion of
PRPP to Histidine
(contd)

Dehydration/
isomerization

Reductive
Amination
NH4+
NADPHNAD+

Phosphate
Hydrolysis

Oxidation

65

David L. Nelson and Michael M. Cox

LEHNINGER
PRINCIPLES OF BIOCHEMISTRY
Sixth Edition

CHAPTER 18

Amino Acid Oxidation and the

Production of Urea
66
2008 W. H. Freeman and Company

Degradation of Proteins
to Amino Acids in the
Stomach

Protease Enzymes
-Gastrin
- Pepsin
- Trypsin
- Chymotrypsin
- Carboxypeptidases A and B

Stomach enzymes
known to degrade
proteins to constituent
amino acids
67

Oxidative Amino Acid Degradation Produces Cellular Energy

and the required building blocks for metabolic pathways
Protein Catabolism
protein

Amino acids

CHO + NH4+

CO2(g) + H2O(l) + ATP

68

Amino Acids will Undergo Oxidative Degradation to ketoacids and a loss of NH4+
Nitrogen Release can occur
in different forms:

69

First Step of Amino Acid Catabolism occurs with loss of NH 4+ in

Transamination Reaction involving Pyridoxal Phosphate (PLP)
Mechansim for transamination

70

PLP acts as a co-enzyme and is readily functionalized by Lys

side-chains in aminotransferases
Mechanism for PLP functionalization of
aminotransferase =

Aminotransferase Enzyme

71

I. Mechanism for PLP-aminotransferase catabolism of amino acids

Overall Reaction :

Mechanism:

72

Glutamate Metabolism serves as a Source of NH4+ in the bloodstream

Phosphorylation

Amminolysis

Hydrolysis

73

Glucose-Alanine Metabolism serves as a Source of NH 4+ in muscle

74

Glu Metabolism and entry into the Urea Cycle

75

Conversion of Glu Arg and its metabolism in the

Formation of Urea

AMP
functionalization
Hydrolysis

Elimination
Asp
functionalization
76

Amino Acid Metabolism Links the Urea and Citric Acid Cycle
Krebs Cycle Summary:
Proteins are digested to their amino acid constituents by proteases. Amino
acids are then metabolized to keto-acids with the loss of ammonia, urea and
urilic acids which undergo further metabolic transformations as part of the
urea cycle. Keto-acids will then undergo further metabolism in the Citric Acid
cycle to form ketones and sugars or complete degradation to its constituent
carbon dioxide and water producing energy for cell.

77

General Overview of Amino Acid Catabolism Involved in the

Citric Acid Cycle

Glucogenic: Amino acids which are converted to glucose (glycogen pathway)

78
Ketogenic: Amino acids which are converted to ketones (gluconeogenesis)

Unregulated Amino Acid Metabolism Leads to Genetic Disorders

For more info: http://www.merck.com/mmpe/sec19/ch296/ch296c.html#

Some Examples:
A. Phenylketonuria (PKU)
Intellectual Stability Disorder caused by inefficient Phe metabolism
Treatment: life-long dietary Phe restriction

B. Tyrosenimia
Liver failure may occur when Enzymes deficient in Tyr metabolism
Treatment: dietary Phe & Tyr restriction if non-effective may lead to liver
transplant

C. Classic homocysteineuria
Detachment of connective tissue results from homocysteine accumulation due to
Enzymes deficient in Met metabolism
79

Treatment: low Met diet, enzyme injections