Download CH 2

32
1
2nd semester
An Introduction to Biochemistry
Bioorganic Chemistry
• 1. Amino acids, Protein and Enzymes
• 2. Other biomolecules:
• Carbohydrates
• Lipids
• Nucleotides and Nucleic Acids
• Vitamins and Coenzymes
Biochemistry - Bioenergetics
• 1. High energy phosphate compounds
• 2. Metabolic Pathways:
• Glycolysis
• Citrate cycle
• Mitochondrial respiration
32
2
Books and Notes
Lehninger: Biochemistry (selected chapters)
Calculation book (previous semester)
Practice book (previous semester)
Collected STUCTURES
You can download the lecture material from our website
www.biokemia.sote.hu
Username: file
Password: open2
32
3
2 Midterms during the semester
Assay questions (topics), calculations, structures
Chemistry Final
2nd semester: 30 multiplichoice questions
1st semester: 20 multiplichoice questions
10 structures
32
4
DR. M. Sasvári
Biochemistry Lectures
Amino Acids, Proteins, Enzymes
Amino Acids
The Building Blocks of Proteins
1. Neutral amino acids
(one amino- and one carboxyl group)
32
5
a-ketoacid derivatives
of amino acids
Amino Acids
+H
3N
COO-
COO-
C H
C
R
R
O
Grouping of amino acids is based on the side chains:
Nonpolar (hydrophobic) – aliphatic and aromatic side chains (only C,H)
Polar, uncharged (hydrophylic) – other atoms (O,N,S) than C and H
Polar, charged (ionic) – weak acids or bases
32
6
Nonpolar (hydrophobic), aliphatic side chains
L-Alanine (3C)
Ala, A
COO+H
3N
C H
CH3
Main metabolic intermediers:
Transamination:
a-ketoacid
COOred
C O
ox
CH3
pyruvate
32
Reduction:
a-hydroxy-acid
COOHO
H
C
CH3
lactate
7
Nonpolar (hydrophobic), aliphatic side chains
BRANCHED CHAIN AMINO ACIDS
Val, V
Valine
Leu, L
Leucine
COO+H
C H
3N
CH
CH3
CH3
COO+H
3N
C
H
CH2
CH
CH3 CH3
Transaminated forms:
aketo  isovalerate
Ile, I
Isoleucine
COO-
aketo  isocapronate
+H
3N
C
H
CH CH3
CH2
CH3
aketo  isocapronate
Metabolic disorder in the catabolism of branched chain amino acids:
32
a-hydroxy-isovalerate and a-hydroxy-isocapronate
in the urine
8
Val
Leu
Ile
32
9
Neutral Amino Acids:
Acid-base character
32
10
Protonated forms
Deprotonated forms
a-carboxyl group is an ACID:
HA
H
pKa1
A +
+
H
-
a-amino group is a base:
B + H+
+
B
H+
pH < pKa
pKa2
32
pKa < pH
11
Neutral Amino Acids:
Protonic equilibria
32
12
H+
pH < pKa1
H
H
+H N
3
C
COOH
R
Form I. Fully
protonated form
pKa1= 2.3
H+
-
H
+H
3N
C
COO-
R
pKa2 = 9.6
-
pH =(pKa1+ pKa2)/2
H
H2 N
C
Form 2.
Isoelectric form
COO-
R
32
Form 3. Fully
deprotonated form
pH > pKa2
13
H
+H
3N
C
BUFFERS
COOH
R
pH around pKa1
Form 2./ Form 1.
BUFFER
pKa1= 2.3
H
+H
3N
C
COO-
NO
Buffer at
Isoelectric pH!
R
pKa2 = 9.6
pH around pKa2
Form 3./ Form 2.
BUFFER
H
H2 N
C
R
COO32
14
Neutral Amino Acids:
pH dependence
of different forms
32
15
Different forms of Ala
mmol
10
5
2
4
6
H
+H
3N
C
8
10
12
H
COOH
+H
3N
C
pH
H
COO-
H2 N
C
R
R
R
+
+ 32 -
-
COO-
16
Neutral amino acids: Titration curve
START:
10 mmol
completely protonated
neutral amino acid
pH
14
12
10
8
Ip
6
4
2
0
0
2
4
6
Buffer 1.
8
10
12
14
16
18
Buffer 2.
1st Eqv.Point32
20
21
meqv NaOH added
2nd Eqv.Point
17
10 ml of 0.1 M glycine solution (completely protonated)
+ 4 ml 0.1 N NaOH, pH=?
pH = 2.3 + lg (deprot./prot.)
pH = 2.3 + lg 4/6 = 2.1
10 ml of 0.1 M completely protonated glycine solution
+ x ml 0.1 N NaOH, pH=2.6
pH = 2.3 + lg x/(10-x) = 2.6
x= 6.66 ml
See also: Selected Calc.
CHAPTER 8. AMINO ACIDS AS BUFFER
32
18
Glycine and Proline: Two extremities
Gly, G
Glycine
Pro, P
Proline
COO+H
3N
C
COO-
H
+H
H
No side chain
maximal flexibility
Aliphatic primary amine
2N
Alicyclic side chain (nonpolar)
rigid
Aliphatic secondary amine
32
19
Gly
Pro
32
20
AROMATIC AMINO ACIDS
hydrophobic interactions between stacking aromatic rings
Phe, F
Phenylalanine
COO+H
3N C
Tyr, Y
Tyrosine
COO+H
H
CH2
3N
Phe hydroxylase
C
H
CH2
(deficiency: phenylketonuria)
nonpolar
Transaminated forms:
phenyl-pyruvate
polar
Phenolic -OH
(very weak acid)
O
H
p-hydroxyphenyl-pyruvate
32
Phenylketonuria: phenyl-pyruvate, phenyl-lactate
and phenyl-acetate in the21urine
AROMATIC AMINO ACIDS
hydrophobic interactions between stacking aromatic rings
Trp, W
Tryptophan
COO+H
3N
C
H
CH2
polar
N
H
Aromatic
secondary amine
in the indol ring
Very weak base
32
22
Aromatic amino acids have a
characteristic absorbance
at 280 nm
Characteristic absorbance of
proteins.
32
23
AROMATIC AMINO ACIDS
with positively charged side chain
His, H
Histidine
COO+H
3N
C
H
CH2
CH2
HN
NH+
aromatic imine
(weak base)
in the imidasol ring
32
HN
+
NH
delocalized structure
24
Ile
Phe
32
Tyr
25
Amino Acids: Optical Activity
Assymetrical (chiral) C atom (4 different substituents)
Enantiomer pairs (mirror images, not imposable on each other)
Optical activity
32
26
Amino Acids: Optical Activity
Proteins Consist of L-Amino Acids
CHO
H
C
CHO
OH
OH
CH2OH
C
H
CH2OH
D-Glyceraldehyde
L-Glyceraldehyde
32
27
Amino Acids: Optical Activity
Proteins Consist of L-Amino Acids
COOH
C
COOH2 N
NH2
C
H
CH3
CH3
D-Alanine
L-Alanine
32
28
32
29
D- Alanine
L- Alanine
32
30
Amino Acids: Optical activity
Some amino acids have 2 chiral centers
COO3N
H
C
H
H
C
NH3+
C
OH
OH
C
H
Diastereomers
CH3
CH3
L-Thr
D-Thr
Diastereomers
+H
COO-
Enantiomer pairs
COO-
COO+H
3N
OH
C
C
H
H
CH3
L-allothreonine
32
H
C
NH3+
H
C
OH
CH3
D-allothreonine
31
Amino Acids: Polar side chains
-OH containing side chains
Thr, T
Threonine
Ser, S
Serine
COO-
COO+H
3N C
+H
H
3N
C
H
CH OH
CH2 OH
CH3
Secondary alcohol
Primary alcohol
32
32
Reactions of the Ser OH group
An ALCOHOL reacting with CARBOXYLIC ACID forms ESTERS
Chemical reaction:
O
R1
C
O
OH
+
H+
R2 OH
Carboxylic acid
heat
Alcohol
R1
C
O
R2 + H2O
Ester
Biochemical reaction:
O
R1
P
O
OH
+
R2 OH
R1
P
O
R2 + H2O
enzyme
OH
OH
Phosphate group
of ATP
Ser –OH
of a protein
Phosphate ester
on a protein
32
33
Thr
COO+H
3N
C
H
CH OH
CH3
32
34
Amino Acids: Grouping principles
-S containing side chains
S: low electronnegativity  slight polarity
Met, M
Methionine
COO-
Cys, C
Cysteine
COO+H
3N C
+H
H
3N
C H
CH2
CH2
S
CH3
CH2 SH
Thioalcohol
32
Thioether
35
Reactions of Amino Acids
Formation of disulfide bridge
Two cysteine units form a disulfide bridge
32
36
A schematic diagram of two disulfides from the protein structure of bovine insulin
32
37
Role of Methionin in the metabolism
Its activated form is a methyl group donor
COO-
COO+H
3N C
H
CH2
CH2
S
CH3
+H
3N
C H
COO+H
CH2
CH2
SH
3N
C
H
CH2
SH
Cystein (3C)
Homocystein (4C)
Methionin (5C)
32
38
Exchange of the sulfur in the metabolism
Homoserine (4C and -OH)
Cystein (3C and -SH)
COO+H
3N
C H
CH2
CH2
COO+H
S
Homocystein (4C and -SH)
32
3N
C
H
Cystathionine
CH2
Serine (3C and -OH)
39
Amino Acids: Polar side chains
Negatively charged (ACIDIC) side chains.
Asp, D
Aspartate
Glu, E
Glutamate
COO+H
3N
COO+H
C H
bCH
C H
CH2
g CH2
2
COO -
COO b-carboxyl
Transamination:
oxaloacetate
3N
32
g-carboxyl
a-keto-glutarate
40
Amino Acids: Polar side chains
Amides
Asn, N
Asparagine
Gln, Q
Glutamine
COO-
COO+H
+H
H
3N C
bCH
3N
C
H
CH2
g CH2
2
CONH2
CONH2
amide
Amide of Aspartate
amide
Amide of Glumatate
32
41
Gln
Glu
COO-
COO+H
3N C
+H
H
3N
C H
CH2
CH2
g CH2
g CH2
COO -
CONH2
32
42
Hydrogen bonds between side chains
Hydrogen donors:
Trp, Ser,Thr,Tyr,Asn, Gln, His
Hydrogen acceptors:
Asp, Glu, Asn, Gln
Side chain
Side chain
Side chain
CH2
C
NH2
CH2
C
NH2
O
O
N
H
H
H
NH
O
Side chain
O
O
C
CH
32 2
Side chain
C O
CH2
43
Side chain
Glu
Tyr
32
44
Gln
Tyr
32
45
Gln
Asn
32
46
Biogen amines
Biochemical reaction: Decarboxylation of amino acids
COO+H
H
3N C
R
Amino acid
CO2
decarboxylation
specific
decarboxylases
(enzymes)
32
+H
3N
C H2
R
Biogen amine
47
Biogen amines
Synthesis of dopamine from Tyr
O2
H2 O
Tyrosine
3N
C H2
CH2
decarboxylation
hydroxylation
Tyr hydroxylase
+H
CO2
Aromatic amino acid
decarboxylase
OH
Dopa
OH
OH
Dopamine
Biogen amine
Dopamine: neurotransmitter
Parkinson’s disease
- low dopamine production
32
48
Biogen amines
Synthesis of norepinephrine and epinephrine
+H
3N
CH3
C H2
O2
H2O +H3N C H2
CH2
HO CH
OH
dopamine
Dopamine
b hydroxylase
vitamin C
methyl group donor
2N
C H2
HO CH
methylation
hydroxylation
OH
+H
OH
OH
OH
phenylethanol amineOH
OH
N-methyl transferase
Norepinephrine
(noradrenaline)
Biogen amine
Neurotransmitter
32
OH
Epinephrine
(adrenaline)
Biogen amine
Stress hormone
49
Biogen amines
Synthesis of GABA from Glu
CO2
H
+H N-C-COO3
CH2
decarboxylation
CH2
Glu
COOdecarboxylase
+H
3N-CH2
CH2
CH2
COO-
Gamma-amino butirate
(GABA)
biogen amine
neurotransmitter
Glu
32
50
Biogen amines
Synthesis of histamine from His
CO2
H
+
NH3 C
COO-
CH2
HN
+
decarboxylation
N
H
His
decarboxylase
His
NH3 C
H
CH2
HN
N
histamine
biogen amine
Vasodilator
Released in allergic response
Stimulation of acid secretion in the stomach
32
51
Biogen amines
Synthesis of serotonin from Trp
COO +H N
3
O2
H2 O
+H N
3
H
C
COO -
CO2
+H N
3
H
C
CH 2
CH2
NH
Trp
CH2
OH
hydroxylation
C H2
OH
decarboxylation
NH
NH
Trp
Aromatic amino acid
decarboxylase
hydroxylase 5-hydroxy-Trp
Serotonin
biogen amine
Neurotransmitter (brain)
Blood coagulation (trombocytes)
32
52
Acidic Amino Acids
Comparison of acidity of different groups
Acidic amino acids
(negative charge at pH 7)
Side chains capable for proton dissociation
(no charge on side chain at pH 7)
Tyr
H
Glu
Asp
H
H
+H N
3
C
COO-
CH2
a
+H N
3
CH2
COO-
b
COO-
C COOCH2
+H N
3
a
C COOCH2
+H N
3
g
4
gcarboxyl
bcarboxyl
acarboxyl
Ser
H
10
thiol
phenol
32
+H N
3
C
CH2
phenol
7
H
COO-
SH
OH
pKa values in different groups:
2
Cys
C COOCH22
OH
thiol
alcohol
14
alcohol
53
Acidic amino acids: Titration curve
Titration of 10 ml 1 M Glu (10 mmol) solution with 1 M NaOH:
Glu : 10ml x 1 M = 10 mmol NaOH: x ml * 1 M = x meqv
pH14
12
Acidic amino acids
have acidic Ip!
10
8
6
4
Ip
2
0
0
2
4
6
8
1st BUFFER
10 12 14 16 18 20 22 24 26 28 30 31
2nd BUFFER
1st Eqv.Point
NaOH ml/meqv added
3rd BUFFER
2nd Eqv.Point
323rd Eqv.Point
54
5
Acidic amino acids: Calculations
Titration of 10 ml 1 M Glu solution with 1 M NaOH:
Glu : 10ml x 1 M = 10 mmol NaOH: (x) ml x 1 M = (x) meqv
NaOH
added
meqv
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
Form 1. Form 2.
Form 3.
mmol
10
8
6
4
2
mmol
0
2
4
6
8
mmol
0
10
8
6
4
2
0
0
2
4
6
8
10
8
6
4
2
0
Form 4.
Calculation of pH
pH
Buffer 1.
1.6
2.0
2.4
2.8
mmol
pH = 2.2 + lg(Form2./Form1.)
Ip:
pH = (pKa1 +pKa2 )/2
Buffer 2.
pH = 4.3 + lg(Form3./Form2.)
0
2
4
6
8
10
pH = (pKa2 +pK a3 )/2
Buffer 3.
pH = 9.7 + lg(Form4./Form3.)
32
3.2
3.6
4.2
4.6
4.9
7.0
9.1
9.5
9.8
10.3
START:
Form 1.
a - COOH
g - COOH
a - NH3+
Form 2.
a - COO g - COOH
a - NH3+
Ip
Form 3.
a - COO g - COO a - NH3+
Form 4.
a - COO g - COO -
a - NH2
55
Amino Acids: Grouping principles
Positively charged (BASIC) side chains.
Lys, K
Lysine
Arg, R
Arginine
COO+H
3N
C H
CH2
CH2
CH2
 CH
2
NH3+
His, H
Histidine
COO+H
3N
C H
CH2
CH2
 CH
2
COO+H
3N
C
H
CH2
HN
NH+
NH
C NH2+
Guanidino
group NH2
32
56
Delocalization of electrons in Arg and His side chains
CH2
HN
Arg: guanidino group
+
NH
His: imidasol ring
32
57
Arg
Lys
32
58
His
32
59
Basic amino acids: Comparison of basicity.
His
Lys
Arg
H
H
H
+NH
3
C
C
NH2
COO-
CH2
HN

N
NH2
COO-
CH2
CH2
CH2
CH2

CH2
COO-
CH2
NH
CH2
C
NH3+
imidasol ring
aryl imine
C
NH2+
NH
 - amino
alkyl amine
  guanidino
alkyl imine
pKa values of different groups:
7
imidasol
10
12
14
a -amino
 -amino
32
alkyl imine
60
Isoelectric forms of basic amino acids
H
+NH
3
C
COO-
CH2
His
HN
Ip: No charge at the side chain
H
H
C
NH2

Lys
N
COO-
NH2
C
CH2
CH2
CH2
CH2
CH2

COO-
CH2
CH2
NH
NH3+
C
NH2+
NH
Ip: Positively charged
side chains
32
Arg
61
Basic amino acids: Titration curve
Titration of 10 ml 1 M Arg (10 mmol) solution with 1 M NaOH:
Arg : 10ml x 1 M = 10 meqv NaOH: (x) ml x 1 M = (x) meqv
pH
14
12
Basic amino acids
have basic Ip!
Ip
10
8
6
4
2
0
0
2
4
6
1st BUFFER
8
10
12
14
16
18
20
2nd BUFFER
1st Eqv.Point
22
24
26
28
30
NaOH ml/meqv added
3rd BUFFER
2nd Eqv.Point
3rd Eqv.Point
32
62
Basic amino acids: Calculations
Titration of 10 ml 1 M Arg solution with 1 M NaOH:
Arg : 10ml x 1 M = 10 mmol
NaOH: x ml x 1 M = x meqv
NaOH Form 1. Form 2. Form 3. Form 4.
added
Calculation of pH
meqv
mmol
mmol
mmol
mmol
0
10
0
2
8
2
Buffer 1.
4
6
4
6
4
6
pH = 2.2 + lg(Form2./Form1.)
8
2
8
10
0
10
0
pH = (pKa1 +pKa2 )/2
12
8
2
Buffer 2.
14
6
4
16
4
6
pH = 9.0 + lg(Form3./Form2.)
18
2
8
20
0
10
0
Ip: pH = (pKa2 +pK a3 )/2
22
8
2
Buffer 3.
24
6
4
26
4
6
pH = 12.5 + lg(Form4./Form3.)
28
2
8
30
0
10
32
pH
1.6
2.0
2.4
2.8
5.6
8.4
8.9
9.3
9.6
10.8
11.9
12.3
12.7
13.1
START:
Form 1.
a - COOH
a - NH3+
NH2+
Form 2.
a - COO a - NH3+
NH2+
Form 3.
a - COO a - NH2
NH2+
Form 4.
a - COO a - NH2
NH
Ip
63
Reactions of Amino Acids
ACYLATION of AMINES forms AMIDES
Chemical reaction:
O
R1
C
R2NH2
Cl
base
O
R1
Acyl chloride
C
NH – R2 + HCl
Amide
Biochemical reaction:
O
R
C
OH
NH3
or amino group donor
R
ATP (energy)
enzyme
O
C
NH2
+ H2O
Amide
e.g. glutamine
Carboxylic acid
e.g. glutamate
32
64
Reactions of Amino Acids
Biochemical reaction: Transamination
An exchange of amino and oxo groups
COO-
oxaloacetate
COO+H
O C
bCH
3N
C
bCH
2
H
Asp
2
COO -
COO -
Aspartate amino transferase (ASAT)
COO-
COO-
Glu
+H
3N
C
O C
H
CH2
CH2
g CH2
COO -
a-ketoglutarate
g CH2
32
COO -
65
Reactions of Amino Acids
A color reaction for amino acids: ninhydrin test
Chemical reaction (amino acid will be broken):
Usage: staining of amino acids and proteins (see: practice)
32
66
Reactions of Amino Acids
A color reaction for amino acids: Sanger’s reagent
Chemical reaction (amino acid will NOT be broken):
HOOC
H
C
R
NH2 + F
NO2
HOOC
NO2
H
C
R
NH
NO2
+HF
NO2
2,4-dinitrofluorobenzene (DNFB)
2,4-dinitrophenylamino acid
(DNP-derivative)
DNP will be attached to amino groups in a protein or peptide
Usage: staining of N-terminal amino acid of proteins (protein sequencing)
32
67
Peptides: N terminal and C terminal
N-terminal
C-terminal
32
68
Reactions of Amino Acids
Chemical reaction Fluorescence labeling of amino acids/proteins
Fluorescence - "glow in the dark".
Usage of fluorescamine:
- sensitive quantification of proteins
- sensitive detection of proteins
32
69
Tubulin in a live bovine pulmonary artery endothelial cell
labeled with green fluorescent reagent
endoplasmic reticulum was visualized
with red-fluorescent reagent
32
70