LEHNINGER PRINCIPLES OF BIOCHEMISTRY th Edition David L. Nelson and Michael M. Cox © 2008 W. H. Freeman and Company CHAPTER 3 Amino Acids, Peptides, and Proteins 1
Dec 20, 2015
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
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
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
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
“NH4+”
NADPH NADP
Biosynthesis of Aspartate from Oxaloacetate
Provide the Intermediate for this Transformation ?42
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)
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
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
Glutamate Metabolism serves as a Source of NH4+ in the bloodstream
Phosphorylation
Amminolysis
Hydrolysis
73
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