CHAPTER 3

PROTEINS
 Ever heard of proteins?
• In Greek, it means “first place” or “of first
importance”
• Natural polymer in our body
• Proteins are constructed in the body from
many repeating units call amino acids
linked by peptide bonds
• Have diverse functions
Proteins are linear polymers of amino acids
Key structural features:
• A "peptide bond" is formed by a
condensation reaction between the
carboxylic acid of one amino acid with
the amino group of the next amino acid
• The amino acid R1, at the "amino
terminus" of the polymer is the "first"
amino acid. The residue (R3 in the
above diagram) at the carboxyl terminal
is known as the "last" amino acid.
These termini define the directionality
of the protein.
Amino acids:
•are organic molecules with both
carboxyl group (-COOH) and amino
group (-NH2)
•have an asymmetric carbon called α-
carbon at the center
•also have different side chain, denoted
as R group
Amino acids:
•are the building blocks of proteins or
•are the monomer or smaller subunit of
proteins
•have 20 common types
•Contains two types of major functional
groups
Amino acids look like this

Carboxyl and amino group in ionized forms

(zwitterion)
Essential amino acids
•Valine •Phenylalanine
•Leucine •Tryptophan
•Isoleucine •Arginine
•Threonine •Histidine
•Lysine
•Methionine
Non-essential amino acids
•Alanine •Glutamine
•Asparagine •Glycine
•Aspartate •Proline
•Cysteine •Serine
•Glutamate •Tyrosine
 Isoeletric point
• Isoecletric point
is the pH at which
the amino acid
net electrical
charge is equal to
zero, and thus
cannot move in
an electrical field.
 Why isoelectric point is important?
We can use these differences in physical properties to fractionate
complex mixtures of amino acids into individual amino acids
•In looking at the isoelectric point of the different amino acids it seems that
they will have different partial charges at a given pH.
•For example, at pH 6.0 some will be negatively charged, and some
positively charged.
•For those that are negatively charged, some will be slightly negative, and
others strongly negative. Similarly, for those that are positively charged,
some will be slightly positive, and others strongly positive
•The charge differences of the amino acids means that they will have
different affinities for other cationic or anionic charges
Ion Exchange Chromatography
• Imagineif we had a tube whose surfaces were
coated with an immobilized cation. These would
have electrostatic attraction for anions.
• If
a solution containing a mixture of positively and
negatively charged groups flows through this tube,
the anions would preferentially bind, and the cations
in the solution would flow through
This is the basis of ion exchange chromatography. The example above is termed "anion
exchange" because the inert surface is interacting with anions If the immobile surface was
coated with anions, then the chromatography would be termed "cation exchange"
chromatography (and cations would selectively bind and be removed from the solution flowing
through Strength of binding can be affected by pH, and salt concentration of the buffer. The
ionic species "stuck" to the column can be removed (i.e. "eluted") and collected by changing
one of these conditions. For example, we could lower the pH of the buffer and protonate anions.
This would eliminate their electrostatic attraction to the immobilized cation surface. Or, we could
increase the salt concentration of the buffer, the anions in the salt would "compete off" bound
anions on the cation surface.
 Zwitterions
• An acid -COOH and
an amine -NH2 group
cannot coexist
• The H+ migrates to the
-NH2 group
• COO- and NH3+ are
actually present, called
a “Zwitterion”
 Zwitterions
• Zwitterion = compound where both a
positive charge and a negative charge
exist on the same molecule
• AA are ionic compounds
• They are internal salts
• In solution their form changes
depending on the pH
AA’s
Zwitterionic structure is neutral and its value of pH is
called isoelectric point
 Amino acids are amphoteric
• That means the amino acids can act as an
acid or a base
• Carboxyl group is acidic
• Amino group is basic
• So can act either as base (proton acceptor) or
an acid (proton donor)
Do not get confused with the acidity and
basicity of the R group
 Amino acids as biosynthetic
precursors
• Many biologically important molecules are
derivatives of amino acids.
• E.g. Tyrosine is the precursor of the hormone
thyroxine, skin pigment melanin and a
compound, DOPA (dihydroxy-phenylalanine),
a neurotransmitter, i.e. transmission of
impulses in the nervous system.
• Tryptophan is the precursor of a vitamin
named nicotinic acid (B3).
 Amino Acid Side Chain
•Amino acids are grouped based on the
properties of their side chains (the R
group)
•The side chain is what makes each AA
unique
•Thus, AA can be categorized into 4
groups
The 20 types of AA
 The Classes of Amino Acids
• Non-polar (hydrophobic)
•R contains aromatic (ring), aliphatic(non ring)
and/or sulfur groups except Tyrosine
• Polar, neutral (hydrophilic)
•R contains –OH or an amide
• Acidic (hydrophilic)
•R contains –COOH that can donate an H+
• Basic (hydrophilic)
•R contains an N that can accept an H+
Nonpolar (hydrocarbons
and one sulfur-containing
amino acid). Dispersion
forces and hydrophobic
effects predominate in
their interactions. They
cannot H-bond with water
and these side chains
have a characteristic
hydrophobic effect in
water.
(nonpolar) (polar) (nonpolar)
Contain functional groups
that can H-bond with
water and other amino
acids. Include C, H, O, N
and S atoms
Basic AA
Nitrogen containing
bases (e.g. guanidino,
imidazole or amino
groups) with a net
positive charge at
neutral pH. Can serve
as proton donors in
chemical reactions, and
form ionic interactions
Carries positive charge
when pH<6
Acidic AA
Contain a carboxylic acid
functional group with a
negative charge at
neutral pH. Can H-bond
with water, can form ionic
interactions, and can also
serve as nucleophiles or
participate in acid-base
chemistry.
 Learning Check
• Classify the following amino acids as hydrophobic
(nonpolar), hydrophilic (polar, neutral), acidic, or basic:

A. Lysine (polar
basic) B. Leucine
(nonpolar)

C. Serine (polar D. Aspartate (polar

neutral) acidic)
Uncommon amino acids
In addition to the 20 common amino acids, there are several uncommon ones
found:
• Hydroxylysine and hydroxyproline. These are found in the protein collagen.
Collagen is a fibrous protein made up of three polypeptides that form a
stable assembly, but only if the proline and lysine residues are hydroxylated.
(requires vitamin C for reduction of these amino acids to hydroxy form)
Thyroxine, an iodinated derivative of tyrosine, found in thyroglobulin
(produced by thyroid gland; requires iodine in diet) g-carboxyglutamic acid
(i.e. glutamic acid with two carboxyl groups) found in certain blood clotting
enzymes (requires vitamin K for production)
• N-methyl arginine and n-acetyl lysine. Found in some DNA binding proteins
known as histones
Amino acid derivatives not found in
proteins
• Some amino acids are made that are not intended for
incorporation into proteins, rather they have important
functionalities on their own
• Serotonin (derivative of tryptophan) and g-amino butyric
acid (a derivative of glutamic acid) are both
neurotransmitters
• Histamine (derivative of histidine) involved in allergic
response Adrenaline (derivative of tyrosine) a hormone
Various antibiotics are amino acid derivatives (penicillin)
Today, you will learn...
•Why proteins are important?
•Simulate the 4 structure levels of proteins
 Protein Structure
• What is the monomer for protein?
• Peptide: A moleculeconsisting of 2 or more AA.
Smaller than proteins. Can be categorized as below:
• Dipeptide: A peptide with 2 amino acids that are joined by a
single peptide bond
• Tripeptide: A peptide with 3 amino acids that are joined by
peptide bonds
• Polypeptide: Polymer of amino acids

• Proteinconsists of one or more polypeptides folded

and coiled into specific conformations
Bond formation involves the subsequent
release of a water molecule
"Proteins" may consist of a single polypeptide,
or a complex of two or more polypeptides
• If two identical polypeptides associate, that complex is
termed a "homo-dimer". If two different polypeptides
associate, that complex is termed a "hetero-dimer"
• A complex of three polypeptides is termed a "trimer", four a
"tetramer", then "pentamer", "hexamer", etc.
• Greek letters are used to describe the composition of
polypeptides in a complex assembly. (NOTE: this
nomenclature says nothing about the structure of the
proteins)
• Protein’s 3D structure is determined by the
linear sequence of amino acids
• Protein function depends on both:
• Amino acid content; and
• Amino acid sequence
• Denaturation or inhibition can change protein
structure and thus affecting its function
• Coenzymes and cofactors enhance the
protein’s structure
The many functions of protein
• Structural
• Your skin, bones, hair, nails are made of proteins
• Catalysis
• Enzymes – biological catalyst
• Movement
• Your muscles are made up of actin and myosin
• Transport
• Blood circulation – blood is made up of hemoglobin
• Transport through membranes – membranes have
protein channels controlling the in and out of
substance
The many functions of protein
• Hormones
• Insulin, oxytocin
• Protection
• Antibody (immune system), fibrinogen (blood clotting)
• Storage
• Casein (milk), albumin (eggs)
• Regulation
• Control of gene expression (histones)
There are three very general
categories of proteins in the body
• Globular. These are water soluble, and the polypeptide chain folds up in
such a way as to place the hydrophobic residues within the core region
and removed from solvent exposure. As their name implies, they are more
or less spherical in shape.
• Fibrous. Often long polymeric chains of simple repeating motifs of a small
set of amino acids. Often forming long, intertwined strands of such
polypeptides that result in high tensile strength (e.g. silk). Generally
insoluble. Function in a structural role in biological assemblies.
• Membrane. Found associated with lipid membranes (often bilayers of fatty
acids). Solubility in non-polar membrane environment is achieved by folding
up so as to expose non-polar residues on the surface of the protein.
Thus, membrane proteins are insoluble in aqueous environment, and
require detergent to be solubilized in water.
 The Shapes of Proteins
• Fibrous • Globular
• Insoluble in water • Partly soluble in water
• Mainly for structural • Not for structural purposes
purposes e.g. Tendons, • Most proteins that move
ligaments (collagen and around e.g. albumin, casein
keratin) • Proteins with binding sites
• Contractile proteins in e.g. enzymes, hemoglobin,
movement e.g. muscles membrane receptor sites.
The Shapes of Proteins
 Level of Protein Structure
• Primary (1°) Protein Structure
– linear sequence of amino acids
• Secondary (2°) Protein Structure
– H bonds in the peptide chain backbone
– Repeating patterns ( helix, pleated sheet)
• Tertiary (3°) Protein Structure
– overall conformation/shape of protein
– Non-covalent interactions between the R groups within the protein
• Quaternary (4°) Protein Structure
– interactions between proteins or multichained protein structure
– Interaction between 2 polypeptide chains
Primary Structure
• The primary structure of a
polypeptide is its amino acid
sequence
• The amino acids are connected by
peptide bonds
• The primary structure determines
the folding of the polypeptide to give
a functional protein
• The amino terminal is the "start"
and the carboxyl terminal is the
"end" AA’s
Primary Structure

• A change in the amino acid sequence can cause its function to change
• Sickle cell anemia – only one change in an amino acid – changes the
hemoglobin’s shape and easily lysed.
Secondary Structure
• The secondary structure of
polypeptides consists of:
• Several repeating patterns
• Common patterns include
α-helix and β-pleated sheet
• These structures are stabilized
by localized hydrogen bond
between the carbonyl and N-H
group in the protein chain
Secondary Structure
•

AA’s
Secondary Structure
Β-pleated sheet
• Several protein chains
• Shape maintained by
intramolecular H bonding
and other attractive forces
between chains
• Chains run anti-parallel
and make U turns at ends

AA’s
Secondary Structure
•Random Coils
• Few proteins have
exclusively α-helix or
β-pleated sheet
• Many have non-repeating
sections called:
Random Coils
Tertiary Structure
• The Three dimensional arrangement of every atom in the
molecule
• Includes not just the peptide backbone but the side chains
as well
• These interactions are responsible for the overall folding
of the protein
• This folding defines its function and it’s reactivity
Tertiary Structure
The final 3D structure of a protein, entailing the shaping of a
secondary structure.
The Tertiary structure is formed by the following interactions:
• Covalent Bonds
• Hydrogen Bonding
• Salt Bridges
• Hydrophobic Interactions
• Metal Ion Coordination
• Van der Waals
These bonds are important for the stabilization of the structure
Tertiary Structure – Covalent bonding
• The most common covalent bond in forming the tertiary
structure is the disufide bond
• It is formed from the disulfide interaction of cysteines
• A strong bond

H H H
[O]
2 HS CH2 C COOH HCOO C CH2 S S CH2 C COOH
[H]
NH2 NH2 NH2
cysteine cystine
Tertiary Structure – Hydrogen bonding
• Anytime you have a hydrogen connected to a F, O, of N
– you can get hydrogen bonding
• These interactions can occur on the side chain,
backbone or both
Hydrogen bonding groups include the main chain carbonyl and amide groups as well as
polar side chains. Polar groups exposed on the surface of proteins often have water as
their hydrogen bonding partner. Polar groups within the core region usually form
hydrogen bonds with other groups within the protein. Most proteins include at least some
buried solvent groups, and these hydrogen bond with side chains and/or main chain
groups in the interior of the protein. Mutational studies have shown that elimination of a
hydrogen bonding partner is often destabilizing to a protein structure, and hydrogen
bonds typically contribute about 12kJ/mol in stabilization energy.
Tertiary Structure – Salt bridge
• Salt bridges are due to charged portions of the protein.
• Opposite charges will attract and form ionic bonds
• Some examples are the NH3+ and COO- areas of the
protein
These interactions between oppositely charged ionic side chains are also known as "salt-bridges". The main
chain amino and carboxyl terminal are fully ionized at physiological pH, as are the side chains Asp (-), Glu (-),
Lys (+), and Arg (+). Histidine can also be charged (+) at pH <=6.0. Opposite charge attraction is modulated by
the dielectric constant of the environment. Charge groups on the surface experience a dielectric constant of
78.5 (that of water) and are therefore weaker than those in the hydrophobic core (with a dielectric of ~4). Thus,
any buried electrostatic interactions are quite strong. The presence of ions in solution can also screen
electrostatic charges and weaken them.
Tertiary Structure – Hydrophobic
interaction
• Because the non-polar groups will turn away from the
water and the polar groups toward it, hydrophobic
interactions take place.
• These interactions are strong enough to help define
the overall structure of a protein
A primary driving force for protein folding involves the removal of non-polar side chains from
solvent exposure. This is accomplished by sequestering them within the core region. Related to
this, interior packing (i.e. van der Waals forces) is optimized by appropriate choice of non-polar
side chains in the primary sequence. Exposing non-polar groups to solvent is entropically costly
(due to water clathrate structure), thus, folding of the polypeptide chain so as to sequester nonpolar
sidechains within the core region is "hydrophobically driven". Some non-polar groups are still found

on the surface of folded proteins.

Tertiary Structure – Hydrophobic
interaction
Tertiary Structure – Metal ion
coordination
• Two side chains with the same charge would normally
repel each other
• However, if a metal is placed between them, they will
coordinate to the metal and be connected together.
• These metal coordinations are important in tertiary
structure formation
Tertiary Structure – Van der Waals
Well packed hydrophobic cores of proteins
represent optimized van der Waals interactions
between non-polar residues. Although
individually weak, numerous neighbor
interactions in such central cores can contribute
a significant stabilization to the native structure.
Quarternary Structure
• The structure formed when two or more
polypeptide chains join together,
sometimes with an inorganic component,
to form a protein.
• Highest level of organization
• Determines how
subunit fit together
• Example Hemoglobin
(4 sub chains)
• 2 chains 141 AA
• 2 chains 146 AA
Hemoglobin and Collagen
• Triple Helix of Collagen
• Structural
protein of
connective tissues
• bone, cartilage, tendon
• aorta, skin
• About 30% of human
body’s protein
• Triple helix units = tropocollagen
 Denaturation
• Denaturation
– The process of unfolding of proteins
– Any physical or chemical agent that destroys
the conformation of a protein is said to
“denature” it
– Examples:
• Heat (boil an egg) to gelatin
• Addition of 6M Urea (breaks H bonds)
• Detergents (surface-active agents)
• Reducing agents (break -S-S- bonds)
 Denaturation
• Denaturation
– Examples:
• Acids/Bases/Salts (affect salt bridges)
• Heavy metal ions (Hg2+, Pb2+)
– Some denaturation is reversible
• Urea (6M) then add to H2O
– Some is irreversible
• Hard boiling an egg
 Denaturation
• Denaturation
Enzymes are characterized by three distinctive features:
catalytic power, specificity and regulation

• Catalytic power
Enzymes can increase reaction rates by up to 1014
times in comparison with the non-enzyme catalyzed
rate
• Regulation
Enzymes may be regulated through the interaction with
specific inhibitor or activator molecules. Thus, their
activity is often tightly regulated
•
• Specificity
Perhaps not surprisingly, enzymes are highly specific for the
reaction they catalyze. Often they will have no affect upon the
reaction rates for reactions involving closely related compounds
(they are designed to catalyze the reaction of a specific reactant,
or substrate). The location on the enzyme where the substrate
binds and reacts is called the active site of the enzyme
Also, as part of this specificity, enzyme catalyzed reactions often
do not involve production of any significant concentrations of by-
products other than the desired product
Many enzymes require an additional non-
protein molecule to be catalytically active.

• Many coenzymes, cofactors, are actively involved in the

chemical reactions that the enzyme catalyzes.
• Many cofactors fall under the category of vitamins
• Some cofactors are covalently bound to the enzyme,
others interact in a non-covalent manner
• Enzymes with bound cofactor are called the holo-enzyme
form. Enzymes with cofactors removed are called the
apo-enzyme form
AMINO ACID METABOLISM
AMINO ACID METABOLISM
 Sub-topics
Explain the metabolism of amino acids
• Describe protein degradation
• Describe the transamination reaction
• Explain the reactions of the urea cycle
• Discuss the catabolism of the amino acids
carbon chains to metabolic intermediates of
carbon metabolism
AMINO ACID METABOLISM

Oxidation

Waste or reuse
OVERVIEW OF AMINO ACID METABOLISM

ENVIRONMENT ORGANISM

Bio-
Ingested synthesis Protein
protein
1 2 3

a
AMINO
ACIDS
b
c c
Degradation Purines
Pyrimidines
(required) Porphyrins
Carbon
Nitrogen
skeletons
(ketogenic) (glucogenic)
Used for
Urea
energy pyruvate
acetoacetate α-ketoglutarate
acetyl CoA succinyl-CoA
fumarate
oxaloacetate
OVERVIEW OF AMINO ACID METABOLISM
 AMINO ACID CATABOLISM

• All enzymes are proteins.

ª Proteins are broken down in stomach and small
intestine to constituent amino acids.

ª Amino acids are either used as building blocks or

burned for energy (~10% of our energy needs).

ª Catabolism of amino acids increases

z for use in gluconeogenesis when glucose is unavailable
(e.g., starvation/diabetes)
z when protein content of diet exceeds need for building
blocks
z during times of stress
OVERVIEW OF AMINO ACID METABOLISM

• Proteins constantly undergo turnover.

 AMINO ACID DEGRADATION
• When dietary amino acids in excess for synthesis of
proteins à require the removal of the amino group.
• Result 2 paths à 1. amino group
(transformed to NH+4)
à 2. remaining carbon
skeleton
(Krebs cycle)
AMINO ACID DEGRADATION
Excess amino acids cannot be stored.
Surplus amino acids are used for fuel.
– Carbon skeleton is converted to
Acetyl–CoA
Acetoacetyl–CoA
Pyruvate
Citric acid cycle intermediate
– The amino group nitrogen is converted to urea and
excreted.
Glucose, fatty acids and ketone bodies can be formed
from amino acids.
Deamination (by
transamination)
 The Degradation of Amino Acid

Amino acid are degraded by

Transamination to give
Or

Oxidative
Pyruvate (3C)
Deamination or
i nt
er
m that forms
which removes ed
iat
amino group as es
of
Acetyl CoA
(2C)
NH4+
that forms Urea
CAC
 Transamination Reaction
• In this reaction, amino group is transferred to α-
keto acid (from α-ketoglutarate)
• Enzyme used = aminotransferase (require
pyridoxal phospate cofactor).
• Acceptor of the amino group becomes amino acid
(glutamate);
• Acceptor of carbon skeleton of amino group
becomes α-keto acid.
• Purpose à remove amino groups from various
amino acids and collect them in glutamate.
– Glutamate acts as single source of amino groups for
biosynthesis or excretion.
 AMINOTRANSFERASE COFACTOR

• All aminotransferases have pyridoxal phosphate (PLP) à acts

as intermediate carrier of transferred amino group.

• It accepts amino group from amino acid & donates to α-keto

acid.

• PLP form with aldehyde group; Pyridoxamine phosphate (PMP)

form with amino group.
Transamination Reaction
Transamination reaction
PLP & PMP
 Oxidative Deamination
(Elimination of NH+4)
• The amino group in glutamate is removed as ammonium
ion (nitrogen toxic to cells).
• Catalyzed by glutamate dehydrogenase and the co-
enzymes is either NAD+ or NADP+.
Transport of amino groups as glutamine
• Peripheral tissues send
their amino groups as
glutamine through the
bloodstream to the liver
for processing.
• High concentration of
ammonium ion (NH+4) shift
the equilibrium of the
reaction to the right, thus
lowering the
concentration of α-
To liver via bloodstream
ketoglutarate for CAC.
Oxidative Deamination
(Elimination of NH+4)
Summary
 Reactions Of The Urea Cycle
• Ammonium ion is highly toxic
• NH4+ combines with CO2 à urea à excreted in urine
• Typical adult excrete 25-30 gram urea in urine
• High consumption of protein à toxic
 THE PATHWAY OF CATABOLIZE
AMINO ACIDS
Step 1: Remove amino group
Step 2: Take amino group to liver for nitrogen
excretion
Step 3: Entry into mitochondria
Step 4: Prepare nitrogen to enter urea cycle
Step 5: Urea cycle
 Step 1. Remove amino group
• Transfer of the amino group of an amino acid to an a-keto
acid Þ the original AA is converted to the corresponding
a-keto acid and vice versa:
Step 2: Take amino group to liver for
nitrogen excretion
• Glutamate releases its
amino group as ammonia in
the liver.

• The amino groups from

many of the a-amino acids
are collected in the liver in
the form of the amino group
of L-glutamate molecules.

The glutamate dehydrogenase of

mammalian liver has the unusual capacity
to use either NAD+ or NADP+ as cofactor
 Nitrogen carriers
1. Glutamate
transferres one amino group WITHIN cells:
Aminotransferase → makes glutamate from a-ketogluta-rate
Glutamate dehydrogenase → opposite
2. Glutamine
transferres two amino group BETWEEN cells → releases its amino
group in the liver
3. Alanine
transferres amino group from tissue (muscle) into the liver
SynthAtase = ATP Move within cells

Move between cells

In liver
Step 3: entry of nitrogen to mitochondria
Step 4: prepare nitrogen to enter urea cycle
Step 5: Urea cycle
 WORD MOLECULE
ORANGE Ornithine
COLORED Carbamoyl Phosphate
CAT Citrulline
ALWAYS Aspartate (enters the cycle)

ASK Argininosuccinate
FOR Fumarate (leaves the cycle)

AWESOME Arginine
UMBRELLA Urea (leaves the cycle)
 WORDS ENZYME
CAN Carbamoyl Phosphate Synthetase 1
OUR Ornithine Transcarbamoylase
AUNTS Argininosuccinate Synthetase
AIM Argininosuccinate Lyase
ACCURATELY Arginase 1
 Urea cycle – review
(Sequence of reactions)
• Carbamoyl phosphate formation in mitochondria is a prerequisite for the
urea cycle
– (Carbamoyl phosphate synthetase)
• Citrulline formation from carbamoyl phosphate and ornithine
– (Ornithine transcarbamoylase)
• Aspartate provides the additional nitrogen to form argininosuccinate in
cytosol
– (Argininosuccinate synthase)
• Arginine and fumarate formation
– (Argininosuccinate lyase)
• Hydrolysis of arginine to urea and ornithine
– (Arginase)
 The overall chemical balance of the
biosynthesis of urea
NH3 + CO2 + 2ATP → carbamoyl phosphate + 2ADP + Pi
Carbamoyl phosphate + ornithine → citrulline + Pi
Citrulline + ATP + aspartate → argininosuccinate + AMP + PPi
Argininosuccinate → arginine + fumarate
Arginine → urea + ornithine

Sum: 2NH3 + CO2 + 3ATP g urea + 2ADP + AMP + PPi + 2Pi