Chem 145: Biochemistry I Notes Methionine

M
I. Amino Acids
1. Nonpolar (Hydrophobic) Amino Acids

Proline
P

Phenylalanine
F
Leucine
L

Alanine 2. Polar (Hydrophilic) Amino Acids

A
Glutamine
Q
Valine
V

Tryptophan
W Asparagine
N

Glycine
Isoleucine G
I

Serine
S

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
 Cysteine Lysine ~12.0
C K

Threonine
T

Arginine ~12.0
Tyrosine R
Y

3. Polar (Hydrophilic) Charged

Acidic pKa
(R) pKa (Cα) = ~2.0
Aspartic Acid ~4.0 pKa (Nα) = ~9.0
D
Essential Amino Acids
PVT TIM HALL

Glutamic ~4.0 -Phenylalanine

Acid Note:
-Valine
E Tyrosine is not
-Threonine
-Tryptophan essential
-Isoleucine because:
-Methionine [O]
Basic -Histidine P↔ Y
Histidine ~6.0 -Alanine
H -Leucine
-Lysine

As a human grows into adulthood, the

number of essential AA’s become 8

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
Properties of Amino Acids Note!

Some notes: 1. When the AA is in its fully-

[1] Phosphorylated Amino Acids – act as protonated form, it is always in a
signaling devices or switches that convert cationic form.
amino acids from an active to an inactive 2. Very acidic = fully protonated form
form (pH ≤ 1) for all amino acids (cation)
[2] Several AA’s occur rarely in proteins 3. Very basic = fully deprotonated
[3] Decarboxylation is [O] form (pH ≥ 13) for all amino acids
(anion)
1. AA’s are weak polyprotic acids 4. All zwitterions are neutral; not the
𝐻2 𝐴+ + 𝐻2 𝑂 → 𝐻𝐴0 + 𝐻3 𝑂+ inverse
[𝐻𝐴0 ][𝐻3 𝑂+ ] 5. Isoelectric point – all structures are
𝐾𝑎1 = purely in its zwitterionic form
[𝐻2 𝐴+ ] 𝑝𝐾𝑎1𝛼−𝐶𝑂𝑂𝐻 + 𝑝𝐾𝑎1𝛼−𝑁𝐻3
𝑝𝐼 = 2
• Ka1 – dictates when the compound will
ionize (COOH to COO- and NH3+ to NH2) At physiological pH (pH=7), the net
charge of PLAV WIMF, QNGS CTY
𝐻𝐴0 + 𝐻2 𝑂 → 𝐴− + 𝐻3 𝑂+ is zero always because NH3 will only
[𝐴−][𝐻3 𝑂+ ] be deprotonated at pH=9.
𝐾𝑎2 =
[𝐻𝐴0 ]
PLAVWIMFQNGSCTY, pI = 5.5
D and E, pI = 3
• Consequences of polyprotic acids
K and R, pI = 10.5
o Structure is pH-dependent
H = 7.5 (net charge at pH=7, +1)
General forms of AA’s
Consequences of H having pI=7.5:
−𝐻 + −𝐻 +
𝑐𝑎𝑡𝑖𝑜𝑛 ⇔ 𝑧𝑤𝑖𝑡𝑡𝑒𝑟𝑖𝑜𝑛 ⇔ 𝑎𝑛𝑖𝑜𝑛 -can easily get protonated and
Net -1 0 -1 deprotonated at physiological pH
Charge
pH 1 7 13 2. Buffering action
• Resist sudden changes in pH
Carboxylic group ionizes first before the • Weak acid (AA) + conjugate base
amino group. • +1 unit of its pKa value
-1 at pH = 2
pH 2 cationic form is in equilibrium with the Example:
zwitterionic form (net charge = ½)
0 What is the pH of glutamic acid if its 𝛼-
at pH = 9
pH 9 anionic form is in equilibrium with the
COOH is ¼ dissociated?
+1 zwitterionic form (net charge = -½) pH = pKa + log ([A-]/[HA])
= 2.2 + log (1/3)
Note: A- is the ion-dipole in aqueous sol’n
HA is the dipole-dipole in solution
[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
Pharmacological application: unambiguously in the latter; they have 2
A drug will be soluble in a certain pH if the chiral centers)
ration [A-]/[HA] is > 1 therefore, [A-] > -D and L molecules enantiomers (mirror
[HA] images) they are diastereomers if they are
not mirror images due to the presence of
3. Reactions of Amino Acids multiple chiral centers
• Carboxyl groups are formed from
amides and esters [R,S] absolute configuration
• Amino groups are from Schiff bases* - Kahn-Ingold-Prelog system
and amides o Priority = highest atomic
• Side chains show unique reactivities: number
o Cys residues can form disulfides o H is usually away from you
o Few rxns are specific to a single kind - R-R = S-S (enantiomers)
of side chain - R,S = S,R (enantiomers)
- R-R = S,R (diastereomers)
*Schiff bases - S-S = R,S (diastereomers)

Lysine is usually involved in forming a
Schiff base
Schiff base - common enzymatic intermediates
where an amine, such as the terminal group of a
lysine residue reversibly reacts with an aldehyde
or ketone of a cofactor or substrate [1]
Application: The common enzyme cofactor PLP forms a
Schiff base with a lysine residue and is transaldiminated to
the substrate(s). Similarly, the cofactor retinal forms a
Schiff base in rhodopsins, including human rhodopsin (via
Lysine 296), which is key in the photoreception mechanism
6. Spectroscopic Properties
All amino acids are IR-active due to the
presence of their functional groups
Only aromatic AA’s are UV active
-aromatic – stability, 2n +1, generally
insoluble, sp2 hybridized – planar
AA’s absorb at 250-280 nm
NMR gives spatial connectivity – structural
elucidation
Frequency of occurrence (least W and M)
Techniques in the Separation of AA
1. Ion-exchange (cation- & anion- exchange)

4. Test for presence of Amino Acids Cation exchange:

Ninhydrin (purple = present) except for
proline which gives a yellow color 𝑠𝑎𝑙𝑡 𝑠𝑜𝑙′ 𝑛
𝑚𝑖𝑥𝑡𝑢𝑟𝑒 𝐴,𝐵,𝐶 𝑢𝑠𝑢𝑎𝑙𝑙𝑦 𝑁𝑎𝐶𝑙
→ a→ Na+
5. Stereochemistry of Amino Acids b b
All but glycine are chiral (Gly = 2 H’s) c c
L- amino acids predominate in nature
[D,L] – nomenclature based on a elutes first and is replaced by Na+ or the
glyceraldehyde where D- = CW and L-CCW cation of the salt because it has the least
[R,S] – absolute configuration; superior to affinity for the column (least positive)
[D,L] (Ile and Leu are named

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
examples:
1. Asp, Ser, Lys
Separation based on charge:
Asp = -1
Ser = 0
Lys = +1

D elutes first on a cation-exchange Peptide planes

column, then S, then K In one peptide plane, the degree of
2. A, G freedom is 3 since there are 3 single bonds
Sep’n based on IMFA that are free to rotate
A(R=CH3)= ion-induced
G(R=H2)= ion-dipole

A elutes first because G has stronger

IMF formed with the column
3. A,V
Sep’n based on size n=5
# of peptide planes = n-1 = 4
Alanine is smaller = more polar, # of peptide bonds = n-1 = 4
Valine elutes first
Peptide planes contain the peptide bonds
II. Proteins
-polymers of amino acids Length:
-read from N-terminal to C-terminal ->=>≡
-peptide bonds formed from alpha-C of Partial double bonds are longer than double
amino acid and alpha-N of the other amino bonds
acid C=O and C=N; N is bigger than O so
Residue = amino acid in a protein C=O<C=N
“Oligo” – 2-20 residues
“Poly” - >20 residues Monomeric – one polypeptide chain
Homomultimer – 1 kind
The Peptide Bond Heteromultimer – more than 1 kind
-usually found in trans- configuration
-has partial (40%) double bond character eg. Hemoglobin (Hb) – tetramer
-about 0.133 nm long heterotetramer – 2 alpha and 2 beta
homomultimers (2 dimers)
Note: trans- configuration allows side chain
(R) separation; no steric strain Size:
Proteins are classified according to shape,
(n-1) = peptide bonds solubility, and function
Where: n= number of residues (1) Shape – globular and fibrous

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
Globular – more diverse functions, generally
soluble
Fibrous – structural functions, long, rod-like
shape, generally more stable

Levels of Structure:
1. Primary (10) – final structure that the
protein adapt, sequence held together by
peptide bonds

2. Secondary (20) – IMFA is the major

driving force in structure
- local folding (H-bond is formed between
carbonyl oxygen and amino hydrogen)
-majorly stabilized by H-bonds
-directionality: C=O to N-H
-𝛂 − 𝐡𝐞𝐥𝐢𝐜𝐞𝐬 𝐚𝐧𝐝 𝛃 − 𝐬𝐡𝐞𝐞𝐭𝐬

3. Tertiary (30) – folding of secondary

structure
-overall 3D shape
-IMFA driving force
-all favorable IMF’s in order to form:
compact, folded, stable structure
-allow forms for intermolecular S-S bridges

4. Quaternary (40) – subunit organization

-no quaternary structures for monomeric
proteins
-can form S-S bridges which are either inter-
or intra-molecular
-can for intra- and inter-molecular H-bonds
eg. Collagen Psi and Phi angles
-fibrous
-made up of tropocollagen – triple helix as a
secondary structure

Myoglobin (Mb) – storage protein

Hb – transport protein

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
Steric combinations on psi and phi
Phi = 0o, Psi= 180o
Phi = 180o, Psi=0o
Classes of Secondary Structures:
Phi= 0o, Psi= 0o
- Alpha helix
- Other helices
Helix Handedness
- Beta sheets (compound of beta
strands)
- Tight turns (beta turns/beta bends)
- Beta bulge
Beta sheet – loose alpha helix

Note: side chains are protruding outwards

of a helix (generally hydrophilic interaction
with solvent)
Interaction among side chains may affect
stability.
H-bonds must form in order to form the
structure.
We need at least 8 residues to form an
alpha-helix.

Amino Acid helix breakers

eg. Tropocollagen – 3 left-handed helices
coiled to form a right-handed triple helix
GLYCINE AND PROLINE
-destabilize the helix
Gly – too flexible due to its small nature
Pro – rigidity due to the ring
Alpha helix – intramolecular H-bonding
Beta sheet – intermolecular H-bonding

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
In beta sheets, side chains alternate between
cavities
Pitch – distance between 2 turns of a helix
Stability of beta sheets: side chains must fit
Pitch = (#residues/turn)(rise)
within the cavities in order to have a stable
stacking of beta sheets
Regular alpha-helix = 3.613
3.6 = #residues per turn
(a) Parallel beta sheet – both N to C terminal
13 = # of atoms in an H-bonded loop
-more extensible because the H-
bonds are compacted/folded (same
Note!
with alpha helix and beta helix)
(1) H-bonded loop dictates the width of
the loop
(2) 310 is the thinnest and longest with a
rise of 0.20 (assuming that the
proteins in comparison have the
same number of residues)
(3) Turns – at least 4 residues to form a
turn
(4) Bulge – results from a missed H-
(b) Antiparallel – 1 is C to N, other is N to C
bonding
-more stable than parallel beta sheet
because H-bonding is linear
Supersecondary structures
-building blocks of domains
1. β α β
2. α α
3. β-meander
4. Greek key

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
 Greek key
Domains
-more complex than supersecondary
structures
-They are stable, independently folding,
compact structural units within a protein,
formed by segments of the polypeptide
chain, with relative independent structure
and function distinguishable from other
regions and stabilized through the same kind
of linkages than the tertiary level.
Following this definition, in this
representation of Pyruvate kinase, it is
possible to distinguish three domains:[2]
1. Parallel twisted sheets
2. β barrel
3. α β barrel
4. β helix
[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
Beta barrel motif
It is very interesting in these motifs that
these repetitive structures can be very
different in their primary structure and
they can be present in very different
proteins. Some proteins have no
supersecondary structures.
Note:
(1) Proteins may be composed of a
single domain
(2) Many proteins can have multiple
domains also
Protein domains may be considered as
elementary units of protein structure and
evolution, capable, to some extent, of folding
and functioning autonomously. A domain
sometimes contain motifs, sometimes don’t.
The tertiary structure of many proteins is
built from several domains.
Domain Functions:
Often each domain has a separate function
to perform for the protein, such as:
– Bind a small ligand
– Spanning the plasma membrane
(transmembrane proteins)
– Contain the catalytic site (enzymes)
– DNA-binding (in transcription factors)
 – Providing a surface to bind specifically to -proteins can have more than 2 subunits
another protein (subunits – individual peptides)

Tertiary Structures – compact, stable Isolation and Purification of Proteins

-only multimeric proteins can form a -no one technique
quaternary structure -at the isoelectric point, IMFs that will be
formed are weaker than when charged (ion-
Disulfide bond formation dipole)
[𝑂] -Gel filtration is the same as gel permeation,
𝐶 + 𝐶 ⇔ 𝐶𝑦𝑠𝑡𝑖𝑛𝑒
size exclusion, and molecular sieve
Protein Folding
-dictated by primary structure
Salting in and Solubility
1. Slow Step – adaption of several
conformations until its gets to a structure
near its final structure
2. Fast Step

Molecular Chaperones
-help molecules to fold properly
-increase the rate of correct folding of
nascent

Salting out – if the concentration of the salt

Predictive Algorithms for Protein
Structure
-Chou-Fasman – based on relative
frequencies of occurrences of the different
AA residues
is > 0.1 M, the proteins salt out of the
solution, increasing the ionic strength and
decreasing the solubility of the protein in the
solvent
-this is because the salt is more mobile than
the side chain of the protein in the solution
and it competes with the side chain for ion-
dipole interactions with the solvent
-the decrease in solvation and neutralization
of the repulsive forces allows the protein to
aggregate and precipitate

Quaternary Structure – for multimeric

proteins
-adapt a symmetric pattern for minimal
energy consumption

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
 3. Size-exclusion Chromatography
-if a molecule is too large to enter the pores
on the beads of the column, it is excluded
from the column at an elution volume
-the smaller the volume, the greater the
elution volume

4. Electrophoresis
-based on the movement of ions in an
electrical field
-generally, electrophoresis is carried out not
in free solution but in a support matrix such
as polyacrylamide or agarose, which retards
the movement of molecules according to
Protein Techniques their dimensions relative to the size of the
pores in the matrix
1. Dialysis and Ultracentrifugation -all proteins with low isoelectric points will
Dialysis be more attracted to the anode, thus will be
-a solution of protein is separated from a found nearer the anode
bathing solution by a semipermeable
membrane Analysis of Proteins
-useful for removing small molecules from
macromolecular solutions or for altering the N-terminal Analysis
composition of the protein-containing -Edman degradation – allows sequential
solution identification
Ultracentrifugation -Edman reagent – phenylisothiocyanate
-improvement of the dialysis principle
-filters with pore sizes over the range of C-terminal Analysis
biomolecular dimensions are used to filter -enzymatic approach
solutions to select for molecules in a Carboxypeptidases – cleave from C-termini
particular size range sequentially
-requires high pressures because sizes are Types:
microscopic 1. A – any except Pro, Arg, Lys
-useful for concentrating dilute solutions of 2. B – Arg, Lys
macromolecules 3. C – any
4. Y – any
2. Ion-Exchange Chromatography
-the salt solution (counterion) used to wash Fragmentation of Polypeptide Chains
the column is added in increasing -usually enzymatic; can be done by specific
concentrations to elute the different proteins or nonspecific chemical means
1. Trypsin – Arg, Lys
2. Chymotrypsin – Trp, Tyr, Phe
3. Clostripain – Arg
[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/
4. Lys-C – Lys
5. Staphylococcal protease – D, E
6. CNBr – Met → sulfonium → cyclic
iminolactone

-other chemical means

7. Hydroxylamine (NH2OH)
a. at pH 9 – Asn-Gly
b. at pH 2.5 – Asp-Pro

[1]
- https://en.wikipedia.org/wiki/Schiff_base#Biochemistry
[2]
- https://biochemistryquestions.wordpress.com/2008/10/09/supersecondary-structures-motifs-and-
domains/