LEHNINGER PRINCIPLES OF BIOCHEMISTRY

th Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 3Amino Acids, Peptides, and Proteins

1

Structure, Assembly and Function of Amino Acids

I. Amino Acid(building block monomers)

II. Peptide(linking of amino acids)

III. Protein(linking of peptides)

2

I. Structure of Amino Acids

R/S-Configuration?Stereochemical Relationship?

Functional Group Components:

Carboxylate C-terminus

AmmoniumN-terminal

side-chain

chiral C

-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

3

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

4

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

5

I. Structure of Amino Acids

Amino Acid Classification by Side Chain Functional Groups:

Identify the functional groups:

6

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:

8

I. Structure of Amino AcidsNomenclature and Properties:

9

Post-Synthesis Modifications of Amino Acids Enzymes catalyze amino acid modifications to alter structure and function in

biological processesI. 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 NatureNatural Amino Acids Undergo post-synthesis modifications for specific biological function

Collagen(connective tissues)

Myosin(motor protein)

Prothrombin(blood coagulation)

Elastin(elastic protein)

Glutathione peroxidase (anti-oxidant protein)

12

Collagen(connective tissues)

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

Synthetic Un-natural Amino Acids function as Molecular Probes to Study Protein Structure & Function

For a review: Bioconjugate Chem. 2009, 20, 1281-1295.

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 Science 2003, 302, 646.

Structural Studies in Prenylated GTPases

Metal chelating amino acids in metalloproteins

J. Am. Chem. Soc. 131, 2481.

14

Physical Properties of Amino Acids-Side-chain aromatic chromophores absorb UV lightUV-VIS spectrophotometer

UV spectra

Beer’s law = log Io = cl(Absorption, A) I

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

Use:

15

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

Resolution of a Racemic Mixture of Amino Acids

polarimetry

Specific rotation

obs ee % = 100 * ( [ R ] - [ S ] ) ( [ R ] + [ S ] )

Optical Purity=

= Specific rotation of sample

Specific rotation pure enantiomer

16

• Thin-Layer 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 (cont’d)Separation of Amino Acids by Chromatography

Physical Properties of Amino Acids (cont’d) Separation of Amino Acids by Chromatography

• Ion-Exchange 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.

Amino acids

Am

ino

acid

s

Note: Ion-Pairing buffer (triethylammonium acetate, TEAA) can be used to elute amino acids from stationary phase

Acid/Base Properties of Amino Acids- Weak acid/base properties due to main & side-chain acidic/basic functional groupsI. 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

Which pH will Gly be protonated and non-protonated 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

equalt to HA

HA H+ + A-

Keq = Ka

21

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

24

Chemical Reactivity of Amino Acids

LEHNINGER PRINCIPLES OF BIOCHEMISTRY

Sixth Edition

David L. Nelson and Michael M. Cox

CHAPTER 3Amino Acids, Peptides, and Proteins

Optional Problems: 1-6

25

LEHNINGER PRINCIPLES OF BIOCHEMISTRY

Sixth Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 22Biosynthesis of Amino Acids,

Nucleotides, and Related Molecules

26

Biosynthesis of the 20 Naturally Occuring Amino AcidsI. 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 & cytosol-

prokaryotes)

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

- Amino Acids are synthesized from a metabolic precursor found in :i.Glycolysisii.Citric Acid Cycleiii.Pentose Phosphate PathwayIdentify the Metabolic pathway associated with

each amino acid biosynthetic family ?

28

29

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

30

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

activity

Co-enzyme involved in functional group

transfer

Co-enzyme involved in methyl group

transfer

Co-enzyme involved in redox activity

Co-enzyme involved in Leu metabolism

Enzymes may contain additional elements or functional groups which help catalyze reactions

Metal Ion cofactors help catalyze enzymatic activity

through covalent coordination bonds

Ion Cofactors

(1) Acid-Base Reactions

Reaction Classes in Metabolism

(3) Rearrangement Reactionsi.e. Claisen Rearrangement

(4) Free-radical Reactions

hv or

(5) Functional Group Transfer Reactions

(6) Oxidation and Reduction Reactions

(7) Pericyclic Reactions

2 NaOH + H2SO4 → 2 H2O + Na2SO4

(2) C-C Reactions

32

a typical Claisen Condensation Reaction

a typical Reductive Amination Reaction

-Ketoglutarate as Starting Material in the biosynthesis of Glutamate, Glutamine, Proline and Arginine in Bacteria

- Part of the citric acid cycle

33

34

Reductive amination and amminolysis of -ketoglutarate leads to L-Gln via L-Glu intermediate

NADPH NADP

Mechanism ?

Intermolecular Reductive Amination- Identify the co-enzymes for the transformations??

I. Amination II. Reduction

Imine Intermediate

OH

O

O

CO2H

NH3

OH

O

HO

CO2H

NH2

H+

OH

O

HN

CO2H

NADPH-->NADP+ H-

H+

OH

O

H2N

CO2H

-ketoglutarate L-Glu

ATP ADP + POH

OH2N

O OPO32-

- P

+ NH3

OH

OH2N

O NH2

L-Gln

35

Glu is precursor to Pro synthesis

Mechanism

Intramolecular Reductive Amination

H2N

O

HCO2H

H+

NH

HOCO2H

H+

N CO2H

NADPH --> NAD+

H-

H+

NH

CO2H

L-Pro

Phosphorylation/activation

Reduction

36

Glu is precursor to Arg synthesis

PG

Reductive Amination

Amide Hydrolysis

Mechanism

H2N

O

OP

O

O-

O-

carbomyl phosphate

NH2

CH

C

H2C

OH

O

H2C

H2CH2N

ornithine

NH2

CH

C

H2C

OH

O

H2C

H2CHN

L-citruline

H2N

O

H2N CO2H

CO2HAsp

NH2

CH

C

H2C

OH

O

H2C

H2CHNH2N

O-P

P-ADP

NH

HO2C

CO2H

NH2

CH

C

H2C

OH

O

H2C

H2CN

HH2N

N

HO2C

CO2H

H

Araginocuccinate

OP

H+

NH2

CH

C

H2C

OH

O

H2C

H2CN

HH2N

NH

Arginine

HO2C

CO2H

fumarate

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 3-Phosphoglycerate

oxidation

NH4+

NADPH NAD

Phosphate hydrolysis

Ser Gly interconversionprovide a plausible mechanism ?

NADHNAD, CO2, NH4+

Reductive amination

38

39

Ser Gly interconversion mechanism

PLP H

O

OH

CO2HH2NSer

PLP protection - reductive amination

OH

CO2HN

PLP

CO2HN

PLP

glycine imine intermediateisomerizationCO2HN

PLP

H2O

CO2HH2N

Gly

B:

- CH2O (reacts with THF)

PLP H

O

Biosynthesis of Cys from Ser in Mammals

Cys + 1C

Thioether formation

nucleophile ?

Redox reaction

40

Oxaloacetate and Pyruvate as starting materials in the biosynthesis of Nine Amino Acids

41

“NH4+”

NADPH NADP

Biosynthesis of Aspartate from Oxaloacetate

Provide the Intermediate for this Transformation ?42

Conversion of Asp to Lys

phosphorylation

reduction

reduction

Aldol Condensation

43

44

Conversion of Asp to Lys

Mechanism for Aldol Condensation

NH2

CH

CH2C

OH

O

C H

O

Asp semialdehyde

H2C

H

O

CO2-

pyruvateB-

H2C

O

CO2-

H+

NH2

CH

CH2C

OH

O

CH

H2C

OHO

CO2-

Aldol product

Conversion of Asp to Lys (cont’d)

Cyclization/ Dehydration

Mechanism of formation ?

Reduction

Hydrolysis/ Acylation

Reductive Amination

“NH4+”

NADPH NADP

45

Conversion of Asp to Lys (cont’d)

Hydrolysis

Epimerization

Sterechemical Relationship ?Chirality ?

Decarboxylation

“NH4+”

NADPH NADPReductive Amination

46

47

Conversion of Asp to Lys

Mechanism for dihydroxypicolinate synthesis and Lys formation

NH2

CH

CH2C

OH

O

CH

H2C

OH O

CO2-

Aldol product

cyclization and imine formation

N CO2-

OHH

B-

H+

elimination/dehydration

N CO2-

H2O

NH

CO2-

H+HO

-O2C

-O2C

-O2CH+

H2N CO2-O

-O2C-O2C

O

SCoA

Succinate-HN

CO2-O

-O2C

Succinyl CoA

1. reductive amination

2. hydrolysis

( H2O)

NH3, NADPH-->NAD

H2N CO2-H2N

-O2Cdiaminopimelate

N CO2-

dihydroxypicolinate

-O2C

NADPH-->NAD

epimerization/decarboxylation

NH2

CH

CH2C

OH

OH2C

H2C

H2C NH2

L-Lys

H+H-

reduction

imine hydrolysis

acylation

Conversion of Asp to Met

Asp Asp--P Reduction

Acylation

Cys Addn

Mechanism ?

48

Succinate-O

O -Succinylhomoserine-PLP

SH

+H3N CO2-

L-Cys

L-Cystathionine

HN-PLP

CO2-

B:

H+

-eliminationSuccinate-O NH-PLP

CO2-

O -Succinylhomoserine-PLP enamine intermediate

tautomerizationNH-PLP

CO2-

-unsaturated imine intermediate "Michael acceptor"

S

+H3N CO2-

NH-PLP

CO2-

tautomerization

H+

S

+H3N CO2-

N-PLP

CO2-

H2O

hydrolysis

S

+H3N CO2-

NH3+

CO2-

Conversion of Asp to Met (cont’d)

Reduction/elimination

Mechanism ?

Methylation

49

S

N CO2-

Cystathionine-PLP intermediate

L-Homocysteine

L-Met

methyl-folate --> folate "methylation"

CO2-

NH3+

PLP

H

B:

elimination

HS CO2-

NH3+

N CO2-PLP

dehydroalainie-PLP imine

H2O-NH3

O CO2-

pyruvateS CO2

-

NH3+

H+

Conversion of Asp to Thr

Reduction

Phosphorylation

Elimination/Hydrolysis

50Phosphorylation

Reduction

Asp

Asp-P

Asp-CHO

Mechanism ?

2-O3PO

Phosphohomoserine-PLP intermediate

CO2-

N-PLP

elimination

HB:

H+

2-O3PO CO2-

NH-PLP

Phosphohomoserine-PLP enamine intermediate

tautomerization

-PO43-

CO2-

N-PLP

Phosphohomoserine-PLP imine intermediate

H2O

a) double bond and b) imine hydrolysis"

ab

CO2-

NH3+

OH L-Thr

Conversion of pyruvate and -ketobutyrate to Ile and Val

Elimination/Imine hydrolysis

Claisen Condensation

Provide a Mechanism ?

51

52

Conversion of pyruvate and -ketobutyrate to Ile and Val

Mechanism for the Claisen Condensation of pyruvate to -acetolactate?

H3C

O

CO2-

pyruvate

CH2

O

-O2C

pyruvate

H

B-

CH2

O

-O2C

H+

OH

-O2C

keto-isomer enol-isomer

OH

-O2C

O

acetolactate

Conversion of pyruvate and -ketobutyrate to Ile and Val (cont’d)

reduction

reduction53

Conversion of pyruvate and -ketobutyrate to Ile and Val (cont’d)

dehydration

Reductive Amination

“NH4+”

NADPHNAD

54

Keto-isovalerate is an intermediate for the biosynthesis of Leu

Claisen Condensation/ Hydrolysis Mechanism ?

rearrangement

oxidation

Reductive amination

“NH4+”

NADPHNAD

55

56

Conversion of keto-isovalerate to a-isopropylmalate

Claisen Condensation/ Hydrolysis Mechanism

CO2-

O

H2C CoA

H

O

B-

H2C CoA

O

keto-isovalerate

acetyl CoA

OH

CoA

O

isopropylmalate-CoA

Claisen Condensation

H2O

OH

OH

Oisopropylmalate

Hydrolysis

CO2H

CO2H

Phosphoenolpyruvate and Erythrose 4-phosphate as starting materials in the biosynthesis of Tryptophan,

Phenylalanine and Tyrosine- As part of glycolysis

57

Conversion of chorismate to Tyr, Phe

Phosphophenol pyruvate

+Erythrose 4-phosphate

Claisen type Rearrangement

Mechanism ?

Decarboxylation/aromatization

Reductive Amination

58

Mechanism ?

59

Conversion of chorismate to Tyr, Phe

Mechanism for the Claisen Type Rearrangement followed by decarboxylation and aromatization

CO2-

HO H

O CO2-

chorismate

-O2C

HO H

prephenate

O

CO2-

HO

O

-O2C

NAD-->NADH

- CO2

4-hydroxy pyruvate

aromatization

a

a

a

a

NAD-->NADH

- CO2

b

O

-O2C

phenylpyruvate

b

b

b

Claisen Rearrangement

Reductive amination

Conversion of chorismate to Trp

Nucleophilic Substituion/Claisen Rearrangement

Provide Mechanism ?

Nucleophilic Displacement

RearrangementMechanism ?

Cyclization

Nucleophilic Displacement with Ser

Provide Mechanism for last 2 steps

NH3

60

Phosphophenol pyruvate

+Erythrose 4-phosphate

61

CO2-

HO H

O CO2-

chorismate

NH3

CO2-

O CO2-

NH2

H H+B

CO2-

NH2

anthranilate

O

OH OH

P-OH2C

O-P-P

PRPP

O

OH OH

P-OH2C HN

CO2-

OH

OH OH

P-OH2C N

CO2-

H B

imine tautomer

OH

OH OH

P-OH2C HN

CO2-

enamine tautomer

=

CO2-

NH

HO O-P

OH

OH

enol-1-carboxyphenylamino-1-deoxyribulose phosphate

NH

O-P

OH

OH

indole-3-glycerophosphate

H+

NH N=PLP-O2C

N

N=PLP

-O2C

H

H+

BH2O

NH

NH2

-O2CL-Trp

-glyceraldehyde 3-P

H+

indoledehydroalanine PLP-imine

Mechanism to Synthesis of

Trp

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

Carbohydrate Rearrangement

(to be discussed with carbohydrates)

Reductive Amination

64

Conversion of PRPP to Histidine (cont’d)

Reductive Amination

Dehydration/isomerization

Phosphate Hydrolysis

Oxidation

NH4+

NADPHNAD+

65

LEHNINGER PRINCIPLES OF BIOCHEMISTRY

Sixth Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 18Amino Acid Oxidation and the

Production of Urea

66

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

68

Protein Catabolism

protein

Amino acids

CHO + NH4+

CO2(g) + H2O(l) + ATP

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 NH4+ 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

Aminotransferase Enzyme

Mechanism for PLP functionalization of aminotransferase =

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 NH4+ in muscle

74

Glu Metabolism and entry into the Urea Cycle

75

Conversion of Glu Arg and its metabolism in the Formation of Urea

76

AMP functionalization

Aspfunctionalization

Elimination

Hydrolysis

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

78

79

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

Treatment: low Met diet, enzyme injections