Download Chapter 4—Amino Acids Amino Acid Structure Amino Acid

BCH 4053—Summer 2001—Chapter 4 Lecture Notes
Slide 1
Chapter 4—Amino Acids
The building blocks of proteins
Slide 2
Amino Acid Structure
• Alpha amino acids-typical structure
• See Figure 4.1
• Amide bond called peptide bond
• See Figure 4.2
• Amino acids differ in R-group
• Structures of the 20 common amino acids
• See Figure 4.3
(LEARN structures, names, and abbreviations!!)
Slide 3
Amino Acid Stereochemistry
• Natural amino acids are L
• Configuration based on glyceraldehyde
• Fischer projection convention:
O
O
C
C H
HO
C H
CH3
L glyceraldehyde
(l-glyce raldehyde)
H
H
C OH
CH 3
D glyce raldehyde
(d-glycera ldehyde)
Chapter 4, Page 1
Slide 4
Amino Acid Stereochemistry,
con’t
• Cahn, Ingold, Prelog System
•
•
•
•
R, S configuration (Web site explanation)
L glyceraldehyde is S
D glyceraldehyde is R
L alanine is S
• See Figure Page 99
• All amino acids are S except:
• Glycine (no chiral carbon)
• Cysteine (why?)
Slide 5
Amino Acids are Polyprotic
• Neutral Side Chain (See Fig. 4.7)
• Acidic Side Chain (See Fig. 4.8a)
• Basic Side Chain (See Fig. 4.8b)
Slide 6
pK’s of Amino Acid Groups
• See Table 1 for actual values
• Approximate values:
•
•
•
•
•
•
•
Alpha carboxyl ~2
Alpha amino ~9.5
Side chain carboxyl ~4
His side chain ~6
Cys side chain ~8.5
Lys and Tyr side chain ~10
Arg side chain ~12
Chapter 4, Page 2
Slide 7
Structure Effect on pK’s
Demonstrate with structures how
electron withdrawing properties of
neighboring functional groups
affects the pK’s.
• Carboxyl group
•
•
•
•
Alkyl carboxyl ~4.5
Side chain carboxy ~4
Alpha carboxyl ~2
Peptide terminal carboxy ~3
• Amino group
•
•
•
•
Alkyl amino ~10.5
Side chain amino ~10.5
Alpha amino ~9.5
Peptide terminal amino~8.
Slide 8
Isoionic Point
• When Σ neg.charges = Σ pos. charges
• pH = (pKa + pKb)/2 where
• pKa is for protonation of neutral species
• pKb is for deprotonation of neutral species
• Illustration for neutral, acidic and basic
amino acids
• Illustration for peptides
Slide 9
Determining Charge on a Peptide
• Note pK’s of all functional groups
• Calculate charge when totally protonated
• Determine number of dissociable protons,
and charge on each intermediate
• Determine pK’s involving neutral species
• pI is average of these pK’s
• pH>pI, peptide is negatively charged
• pH<pI, peptide is positively charged
Chapter 4, Page 3
Slide
10
Determining Charge on a
Peptide, con’t.
• Consider the peptide ACKRDM
• Estimate its pI
• Estimate its charge at
• pH = 4.0, pH = 7.0, and pH = 10.0
• How to proceed?
• First, determine the protonatable groups and
their pK’s
Slide
11
Functional Groups of ACKRDM
•
•
•
•
•
•
Peptide amino terminal, pK ~ 8
Cysteine side chain, pK ~ 8.5
Lysine side chain, pK ~ 10
Arginine side chain, pK ~ 12
Aspartate side chain, pK ~ 4
Peptide carboxy terminal, pK ~ 3
Slide
12
Charge of Fully Protonated
ACKRDM
Ala
O
Cys
O
Lys
O
Arg
O
Asp
O
Met
O
H3NCHC NHCHC NHCHC NHCHC NHCHC NHCHC OH
o pK~3
CH2
CH3
CH2
CH2
CH2
CH2
pK~8
oSH
CH2
CH2
pK~8.5
CH2
C OH
o
O
CH2
CH2
CH2
NH
pK~4
CH3
NH3
C NH2
pK~10
S
NH2
pK~12
Total charge of fully protonated form = +3
Chapter 4, Page 4
Slide
13
Charge of Fully Unprotonated
ACKRDM
Ala
Cys
Lys
Arg
Asp
Met
O
O
O
O
O
O
o
H2N CHC NHCHC NHCHC NHCHC NHCHC NHCHC O
CH2
CH3
CH2
CH2
CH2
CH2
p K~8
CH2
CH2
C O
CH2
S
CH2
CH2
O
S
CH2
NH
pK~4
CH3
pK~8.5
NH2
o
pK~10
pK~3
C NH
o
NH2
pK~12
Total charge of fully unprotonated form = -3
Slide
14
Dissociation Steps for ACKRDM
pK1
+3
pK2
+2
3
ca rboxyl te rm.
+1
4
pK3
0
8
aspartate amino term.
pK4
-1
pK5
pK6
-2
8.5
10
12
cysteine
lysine
arginine
pI = (pK3 +pK4 )/2
-3
(Average of pK' s of
= (8 + 8.5)/2 = ~8.3
neutral spe cies)
Slide
15
ACKRDM Charge at various pH’s)
Group
pH = 4.0
pH = 7.0
pH = 10.0
Amino Term.
+1
~+0.9
~+0.03
Cysteine
~0
~-0.03
~-0.97
Lysine
+1
+1
~+0.5
Arginine
+1
+1
~+.99
Aspartate
~-0.5
-1
-1
Carbox. Term.
~-0.9
-1
-1
Total
~+1.6
~+0.9
~-1.5
Chapter 4, Page 5
Slide
16
Other Properties of Amino Acids
•
•
•
•
•
Ultraviolet Spectra (See Figure 4.15)
Reactions of Carboxyl Group (See Figure 4.9a)
Reactions of Amino Group (See Figure 4.9b)
Ninhydrin Reaction (See Figure 4.10)
Reactions of Cysteine SH (See Figure 4.11a and
4.11b)
• 13C Chemical Shifts with pH (See Figure 4.17)
Slide
17
Chromatographic Separation of
Amino Acids
• Ion Exchange Chromatography
• Cation Exchange Resin
• Structure (See Figure 4.18a)
• Operation (See Figure 4.19)
• Anion Exchange Resin (See Figure 4.18b)
• Analysis on Ion Exchange
• See Figure 4.21a
• See Figure 4.21b
• HPLC Reversed Phase Chromatography of Chemical
Derivative
• See Figure 4.22
Chapter 4, Page 6