PROTEINS – When present in proteins, they are

located in the interior of protein where

Characteristics of protein
there is no polarity
• A protein is a naturally occurring, unbranched
polymer in which the monomer units are amino acids Polar amino acids: R-groups are polar
• Proteins are most abundant molecules in the cells – Three types: Polar neutral; Polar acidic;
after water – account for about 15% of a cell’s overall and Polar basic
mass • Polar-neutral: contains polar but neutral side
• Elemental composition - Contain Carbon (C), chains
Hydrogen (H), Nitrogen (N), Oxygen (O), most also – Seven amino acids belong to this
contain Sulfur (S) category
• The average nitrogen content of proteins is 15.4% by – Contains SH, OH, CONH2
mass • Polar acidic: Contain carboxyl group as part of
the side chains
• Also present are Iron (Fe), phosphorus (P) and some
– Two carboxyl acids belong to this
other metals in some specialized proteins
category
– Contains COOH
Amino Acid: The building blocks for protein
• Polar basic: Contain amino group as part of the
side chain
• Amino acid - An organic compound that contains
– Two amino acids belong to this category
both an amino (-NH2) and carboxyl (-COOH)
– Contains NH2 or N2
groups attached to same carbon atom
– The position of carbon atom is Alpha (a)
– -NH2 group is attached at alpha (a)
carbon atom.
– -COOH group is attached at alpha (a)
carbon atom.
• R = side chain –vary in size, shape, charge,
acidity, functional groups present, hydrogen-
bonding ability, and chemical reactivity.
– >700 amino acids are known
– Based on common “R” groups, there are
20 standard amino acids

Side Chain

R  -Carbon Atom

H2N C COOH
-Carboxyl
-Amino H
Group
Group

• All amino acids differ from one another by their

R-groups
• Standard amino acids are divided into four groups
based on the properties of R-groups
• Non-polar amino acids: R-groups are non-polar
– Such amino acids are hydrophobic-water
fearing (insoluble in water)
– 8 of the 20 standard amino acids are non
polar (9 because glycine is unique)
Nomenclature of Amino Acids • Chiral centers exhibit enantiomerism (left- and
right-handed forms)
• Common names assigned to the amino acids are • Each of the 19 amino acids exist in left and right
currently used. handed forms
• Three letter abbreviations – widely used for
naming:
- First letter of amino acid name is compulsory
and capitalized followed by next two letters
not capitalized except in the case of
Asparagine (Asn), Glutamine (Gln) and
tryptophan (Trp).
• One-letter symbols – commonly used for
• The amino acids found in nature as well as in proteins
comparing amino acid sequences of proteins:
are L isomers.
- Usually the first letter of the name
– Bacteria do have some D-amino acids
- When more than one amino acid has the same
– With monosaccharides nature favors D-
letter the most abundant amino acid gets the
isomers
1st letter.
• The rules for drawing Fischer projection formulas for
• Both types of abbreviations are given in the
amino acid structures
following slides
• The — COOH group is put at the top, the R group at
O
the bottom to position the carbon chain vertically
O • The — NH2 group is in a horizontal position.
d. H 2N CH C OH
a. H 2N CH C OH – Positioning — NH2 on the left – L isomer
CH2
CH3 – Positioning — NH2 on the right – D
isomer.
O
H 2N CH C OH
CH2
b.
O
H 2N CH C OH
e. CH2
OH
CH2
CH2
O CH2
H 2N CH C OH COOH COOH COOH
c. NH2
CH2 *C *C H 2N *C H
H2N H H NH2
C O
OH CH CH3 CH2 CH2

CH2 SH
Analyze each structure
Answers; a. nonpolar, b polar neutral, c polar acidic, CH3
d non-polar, e. polar basic

Chirality and Amino Acid OH

Analyze each structure
• Four different groups are attached to the a-carbon A = L-Isoleusine
atom in all of the standard amino acids except B = D-Cysteine
glycine C = L-Tyrosine
– In glycine R-group is hydrogen
• Therefore 19 of the 20 standard amino acids
contain a chiral center Acid-Base Properties of Amino Acids
 • In pure form amino acids are white crystalline o The amino acid carries a net positive charge
solids (cationic form).
• Most amino acids decompose before they melt • At neutral pH (around pH 7, physiological pH):
• Not very soluble in water o The carboxyl group (-COO⁻) loses a proton
and becomes negatively charged.
• Exists as Zwitterion: An ion with + (positive) and
o The amino group remains protonated as -
– (Nagetive) charges on the same molecule with
NH₃⁺.
a net zero charge o The amino acid exists in a zwitterionic form
– Carboxyl groups give-up a proton to get (has both a positive and negative charge but
negative charge is overall neutral).
– Amino groups accept a proton to become • At high pH (basic conditions, pH > 9):
positive o The carboxyl group remains deprotonated (-
COO⁻).
COO- COO- o The amino group loses a proton and becomes
-NH₂.
+
H 3N H H NH3 + o The amino acid carries a net negative charge
(anionic form).
2. Isoelectric Point (pI)
R CH3
• The isoelectric point (pI) is the pH at which the amino
L D acid has no net charge (exists as a zwitterion).
• Amino acids in solution exist in three different • For most neutral amino acids, the pI is around 5-6.
species (zwitterions, positive ion, and negative • For acidic amino acids (like aspartic acid and
ion) – Equilibrium shifts with change in pH glutamic acid), the pI is lower (~3-4).
• Isoelectric point (pI) – pH at which the • For basic amino acids (like lysine and arginine), the
pI is higher (~9-11).
concentration of Zwitterion is maximum – net
3. Effect of R Groups on Acid-Base Behavior
charge is zero
• Nonpolar & Neutral Amino Acids (e.g., glycine,
– Different amino acids have different alanine) → Behave as described above.
isoelectric points • Acidic Amino Acids (e.g., aspartic acid, glutamic acid)
– At isoelectric point – amino acids are not → Have extra carboxyl (-COOH) groups, making
attracted towards an applied electric field them more acidic.
because they net zero charge. • Basic Amino Acids (e.g., lysine, arginine, histidine) →
Have extra amino (-NH₂) or imidazole groups, making
COOH COO- COO- them more basic.
+
H3N C H
+
H3N C H H2N C H 4. Titration Curve of Amino Acids
• When titrated with a strong base (like NaOH), amino
CH3 CH3 CH3
High pH
acids show buffering regions around their pKa values.
Low pH Zwitter Ion
(net + charge) (net neutral charge) (net - charge) • The titration curve has two pKa values for simple
amino acids (one for -COOH and one for -NH₃⁺).
Little explanation: • For amino acids with ionizable side chains (e.g.,
The acid-base properties of amino acids are due to their histidine, glutamate, lysine), there is an additional
amphoteric nature, meaning they can act as both acids and third pKa.
bases. This is because each amino acid has at least two
ionizable functional groups:
1. Amino group (-NH₂ or -NH₃⁺) → Can accept a proton Cysteine: A Chemically Unique Amino Acid
(acts as a base)
2. Carboxyl group (-COOH or -COO⁻) → Can donate a • Cysteine: the only standard amino acid with a
proton (acts as an acid) sulfhydryl group ( — SH group).
1. Ionization States of Amino Acids • The sulfhydryl group imparts cysteine a chemical
The charge of an amino acid depends on the pH of the property unique among the standard amino acids.
surrounding environment. Here’s how it behaves at different pH
• Cysteine in the presence of mild oxidizing agents
levels:
dimerizes to form a cystine molecule.
• At low pH (acidic conditions, pH < 2):
o The carboxyl group (-COOH) remains
protonated. Cystine - two cysteine residues linked via a covalent
o The amino group (-NH₃⁺) is also protonated. disulfide bond
 Peptide Nomenclature

• The C-terminal amino acid residue keeps its full

amino acid name.
• All of the other amino acid residues have names that
end in -yl. The -yl suffi x replaces the -ine or -ic acid
ending of the amino acid name, except for
tryptophan, for which -yl is added to the name.
• The amino acid naming sequence begins at the N-
terminal amino acid residue.
• Example:
- Ala-leu-gly has the IUPAC name of
alanylleucylglycine
Peptides
Isomeric peptides
• Under proper conditions, amino acids can bond
together to produce an unbranched chain of amino • Peptides that contain the same amino acids but
acids. present in different order are different molecules
• The length of the amino acid chain can vary from a (constitutional isomers) with different properties
few amino acids to many amino acids. – For example, two different dipeptides can
• Such a chain of covalently linked amino acids is be formed between alanine and glycine
called a peptide. • The number of isomeric peptides possible
• The covalent bonds between amino acids in a increases rapidly as the length of the peptide
peptide are called peptide bonds. chain increases

• Dipeptide: bond between two amino acids

• Oligopeptide: bond between ~ 10 - 20 amino Biochemically Important Small peptides
acids
• Polypeptide: bond between large number of • Many relatively small peptides are biochemically
amino acids active:
• Every peptide has an N-terminal end and a C- – Hormones
terminal end – Neurotransmitters
+
H3N-aa-aa-aa-aa-aa-aa-aa-aa-aa-COO- – Antioxidants
• Small Peptide Hormones:
OH – Best-known peptide hormones: oxytocin
N-terminal end and vasopressin
O O CH2 O
– Produced by the pituitary gland
H H
+H
3N CH C N CH C N CH C O- – nonapeptide (nine amino acid residues)
with six of the residues held in the form of
CH3 CH2 C-terminal end
a loop by a disulfide bond formed between
two cysteine residues

Alanine Phenylalanine Serine

Small Peptide Neurotransmitters – Multimeric: A multimeric protein
contains more than one peptide chain
• Enkephalins are pentapeptide neurotransmitters
produced by the brain and bind receptor within Protein Classification based on Chemical Composition
the brain
• Help reduce pain • Simple proteins: A protein in which only amino
• Best-known enkephalins: acid residues are present:
– Met-enkephalin: Tyr–Gly–Gly–Phe–Met – More than one protein subunit may be
– Leu-enkephalin: Tyr–Gly–Gly–Phe–Leu present but all subunits contain only
amino acids
Small Peptide Antioxidants • Conjugated protein: A protein that has one or
more non-amino acid entities (prosthetic groups)
• Glutathione (Glu–Cys–Gly) – a tripeptide – is present in its structure:
present is in high levels in most cells – One or more polypeptide chains may be
• Regulator of oxidation–reduction reactions. present
• Glutathione is an antioxidant and protects cellular – Non-amino acid components - may be
contents from oxidizing agents such as peroxides organic or inorganic - prosthetic groups
and superoxides – Lipoproteins contain lipid prosthetic
– Highly reactive forms of oxygen often groups
generated within the cell in response to – Glycoproteins contain carbohydrate
bacterial invasion groups,
• Unusual structural feature – Glu is bonded to Cys – Metalloproteins contain a specific metal
through the side-chain carboxyl group. as prosthetic group

4 Types of Structure

– Primary Structure
– Secondary Structure
– Tertiary Structure
– Quaternary
• Primary Structure: Primary structure of protein
General Structural characteristics of proteins refers to the order in which amino acids are linked
together in a protein
• General definition: A protein is a naturally- • Every protein has its own unique amino acid
occurring, unbranched polymer in which the sequence
monomer units are amino acids. – Frederick Sanger (1953) sequenced and
• Specific definition: A protein is a peptide in determined the primary structure for the
which at least 40 amino acid residues are present: first protein - Insulin
– The terms polypeptide and protein are
often used interchangeably used to Primary Structure of a Human Hemoglobin
describe a protein
– Several proteins with >10,000 amino
acid residues are known
– Common proteins contain 400–500
amino acid residues
– Small proteins contain 40–100 amino
acid residues
• More than one peptide chain may be present in a
protein:
– Monomeric : A monomeric protein
contains one peptide chain
 • Proteins of the same organism always same Beta pleated sheets
sequence (cows, pigs, etc.)
• Different sources: Insulin from pigs, cows, sheep, • Completely extended amino acid chains
humans similar • H-bonding between two different chains – inter
• Some differences: and/or intramolecular
• Side chains below or above the axis
Species Chain A Chain B
AA #8 AA #9 AA #10 AA #30
Human Thr Ser Ile Thr
Pig (porcine) Thr Ser Ile Ala
Cow (bovine) Ala Ser Val Ala

• Due to differences insulin may show some

reaction over time
• Now human insulin produced from genetically
engineered bacteria

Secondary Structure of Proteins

• Arrangement of atoms of backbone in space. Tertiary Structure of Proteins

• The two most common types : alpha-helix (a-
helix) and the beta-pleated sheet (b-pleated sheet). • The overall three-dimensional shape of a protein
• The peptide linkages are essentially planar thus • Results from the interactions between amino acid
allows only two possible arrangements for the side chains (R groups) that are widely separated
peptide backbone for the following reasons: from each other.
– For two amino acids linked through a • In general 4 types of interactions are observed.
peptide bond six atoms lie in the same
plane 4 types of interaction
– The planar peptide linkage structure has • Disulfide bond: covalent, strong, between two
considerable rigidity, therefore rotation cysteine groups
of groups about the C–N bond is hindered • Electrostatic interactions: Salt Bridge between
– Cis–trans isomerism is possible about C– charged side chains of acidic and basic amino
N bond. acids
– The trans isomer is the preferred – -OH, -NH2, -COOH, -
orientation CONH2
• H-Bonding between polar, acidic and/or basic R
Alpha Helix groups
• A single protein chain adopts a shape that – For H-bonding to occur, the H must be
resembles a coiled spring (helix): attached on O, N or F
– H-bonding between same amino acid • Hydrophobic interactions: Between non-polar
chains –intra-molecular side chains
– Coiled helical spring
– R-group outside of the helix -- not Quaternary Structure of proteins
enough room for them to stay inside
• Quaternary structure of protein refers to the
organization among the various peptide chains in
a multimeric protein:
• Highest level of protein organization
• Present only in proteins that have 2 or
more polypeptide chains (subunits)
• Subunits are generally Independent of
each other - not covalently bonded
• Proteins with quartenary structure are
often referred to as oligomeric proteins
• Contain even number of subunits
Protein Classification based on shape – Oxygen stored in myoglobin molecules
serves as a reserve oxygen source for
• Three types of proteins: fibrous, globular, and working muscles
membrane
• Fibrous proteins: protein molecules with elongated Globular Proteins: Hemoglobin
shape: • An oxygen carrier molecule in blood
– Generally insoluble in water • Transports oxygen from lungs to tissues
– Single type of secondary structure • Tetramer (four peptide chains) - each subunit has
– Tend to have simple, regular, linear a heme group
structures • Can transport up to 4 oxygen molecules at time
– Tend to aggregate together to form • Iron atom in heme interacts with oxygen
macromolecular structures, e.g., hair, • Proteins play crucial roles in most biochemical
nails, etc processes.
• Globular proteins: protein molecules with peptide • The diversity of functions exhibited by proteins
chains folded into spherical or globular shapes: far exceeds the role of other biochemical
– Generally water soluble – hydrophobic molecules
amino acid residues in the protein core • The functional versatility of proteins stems from:
– Function as enzymes and intracellular • Ability to bind small molecules specifically and
signaling molecules strongly
• Membrane proteins: associated with cell • Ability to bind other proteins and form fiber-like
membranes structures, and
– Insoluble in water – hydrophobic amino • Ability integrated into cell membranes
acid residues on the surface
– Help in transport of molecules across the Protein Classification Based on function
membrane
• Catalytic proteins: Enzymes are best known for their
Fibrous Proteins: Alpha Keratin catalytic role.
• Provide protective coating for organs – Almost every chemical reaction in the
• Major protein constituent of hair, feather, nails, body is driven by an enzyme
horns and turtle shells • Defense proteins: Immunoglobulins or antibodies
• Mainly made of hydrophobic amino acid residues are central to functioning of the body’s immune
• Hardness of keratin depends upon -S-S- bonds system.
• more –S-S– bonds make nail and bones hard • Transport proteins: Bind small biomolecules, e.g.,
oxygen and other ligands, and transport them to
Fibrous Proteins: Collagen other locations in the body and release them on
• Most abundant proteins in humans (30% of total demand.
body protein) • Messenger proteins: transmit signals to coordinate
• Major structural material in tendons, ligaments, biochemical processes between different cells,
blood vessels, and skin tissues, and organs.
• Organic component of bones and teeth – Insulin and glucagon - regulate
• Predominant structure - triple helix carbohydrate metabolism
• Rich in proline (up to 20%) – important to – Human growth hormone – regulate body
maintain structure growth
• Contractile proteins: Necessary for all forms of
Globular Proteins: myoglobin movement.
– An oxygen storage molecule in muscles. - Muscles contain filament-like contractile
– Monomer - single peptide chain with one proteins (actin and myosin).
heme unit - Human reproduction depends on the
– Binds one O2 molecule movement of sperm – possible because of
– Has a higher affinity for oxygen than contractile proteins.
hemoglobin. • Structural proteins: Confer stiffness and rigidity
- Collagen is a component of cartilage a
 - Keratin gives mechanical strength as well as
protective covering to hair, fingernails,
feathers, hooves, etc.
• Transmembrane proteins: Span a cell membrane and
help control the movement of small molecules and
ions.
- Have channels – help molecules can enter and
exist the cell.
- Transport is very selective - allow passage of
one type of molecule or ion. – Egg white - a concentrated solution of
• Storage proteins: Bind (and store) small molecules. protein albumin - forms a jelly when
- Ferritin - an iron-storage protein - saves iron heated because the albumin is denatured
for use in the biosynthesis of new hemoglobin • Cooking:
molecules. – Denatures proteins – Makes it easy for
- Myoglobin - an oxygen-storage protein enzymes in our body to hydrolyze/digest
present in muscle protein
• Regulatory proteins: Often found “embedded” in – Kills microorganisms by denaturation of
the exterior surface of cell membranes - act as sites proteins
for receptor molecules – Fever: >104ºF – the critical enzymes of
- Often the molecules that bind to enzymes the body start getting denatured
(catalytic proteins), thereby turning them
“on” and “off,” and thus controlling Glycoprotein
enzymatic action.
• Nutrient proteins: Particularly important in the early • Conjugated proteins with carbohydrates linked to
stages of life - from embryo to infant. them:
- Casein (milk) and ovalalbumin (egg white) – Many of plasma membrane proteins are
are nutrient proteins glycoproteins
- Milk also provide immunological protection – Blood group markers of the ABO system
for mammalian young. are also glycoproteins
– Collagen and immunoglobulins are
Protein Hydrolysis glycoproteins

• Hydrolysis of proteins - reverse of peptide bond • Collagen -- glycoprotein

formation: – Most abundant protein in human body
- Results in the generation of an amine and a (30% of total body protein)
carboxylic acid functional groups. – Triple helix structure
- Digestion of ingested protein is enzyme- – Rich in 4-hydroxyproline (5%) and 5-
catalyzed hydrolysis hydroxylysine (1%) — derivatives
- Free amino acids produced are absorbed into – Some hydroxylysines are linked to glucose,
the bloodstream and transported to the liver galactose, and their disaccharides – help in
for the synthesis of new proteins. aggregation of collagen fibrils.
- Hydrolysis of cellular proteins and their
resynthesis is a continuous process. Immunoglobulin
• Glycoproteins produced as a protective response
Protein Denaturation to the invasion of microorganisms or foreign
Partial or complete disorganization of molecules - antibodies against antigens.
protein’s tertiary structure • Immunoglobulin bonding to an antigen via
- Cooking food denatures the protein but does variable region of an immunoglobulin occurs
not change protein nutritional value through hydrophobic interactions, dipole – dipole
• Coagulation: Precipitation (denaturation of interactions, and hydrogen bonds.
proteins)
 Enzyme Structure

Simple and Conjugated Enzyme

• Enzymes are of two types: simple enzymes and

conjugated enzymes
• Simple enzyme: composed only of protein
(amino acid chains)
• Conjugated enzyme: Has a nonprotein part in
addition to a protein part.
– Apoenzyme: Protein part of a conjugated
enzyme.
Lipoprotein
– A cofactor : Nonprotein part of a
conjugated enzyme.
• Lipoprotein: a conjugated protein that contains
– A holoenzyme is the biochemically
lipids in addition to amino acids
active conjugated enzyme
• Major function - help suspend lipids and transport
– Apoenzyme + cofactor = holoenzyme
them through the bloodstream
(conjugated enzyme)
• Four major classes of plasma lipoproteins:
– Chylomicrons: Transport dietary
Cofactors
triacylglycerols from intestine to liver
and to adipose tissue.
• Cofactors are important for the chemically
– Very-low-density lipoproteins (VLDL):
reactive enzymes
Transport triacylglycerols synthesized in
• Cofactors are small organic molecules or
the liver to adipose tissue.
Inorganic ions
– Low-density lipoproteins (LDL):
– Organic molecule cofactors: also called
Transport cholesterol synthesized in the
as co-enzymes or co-substrates
liver to cells throughout the body.
– Co-enzymes/co-substrates are derived
– High-density lipoproteins (HDL):
from dietary vitamins
Collect excess cholesterol from body
– Inorganic ion cofactors
tissues and transport it back to the liver
– Typical metal ion cofactors - Zn2+, Mg2+,
for degradation to bile acids.
Mn2+, and Fe2+
– Nonmetallic ion cofactor - Cl-
– Inorganic ion cofactors derived from
ENZYMES AND VITAMINS
dietary minerals
General Characteristics of Enzymes
Nomenclature and Classification of Enzyme
• Enzymes are catalysts and are not consumed in
the reactions
• Nomenclature: Most commonly named with
• Enzymes are proteins that act as a catalyst for
reference to their function
biochemical reactions
– Type of reaction catalyzed
• The human body has 1000s of enzymes
– Identity of the substrate
• Enzymes are the most effective catalysts known
• A substrate is the reactant in an enzyme-catalyzed
• Most enzymes are globular proteins
reaction:
• A few enzymes are now known to be ribonucleic
– The substrate is the substance upon
acids (RNA)
which the enzyme “acts.”
• Enzymes undergo all the reactions of proteins
– E. g., In the fermentation process sugar to
including denaturation
be converted to CO2, therefore in this
• Enzyme activity is dramatically affected by:
reaction sugar is the substrate
– Alterations in pH
– Temperature
– Other protein denaturants
Three Important aspects of the Naming Process – An oxidoreductase requires a coenzyme
that is either oxidized or reduced as the
1. Suffix -ase identifies it as an enzyme substrate in the reaction.
– E.g., urease, sucrase, and lipase are all
enzyme designations – E.g., Lactate dehydrogenase is an
– Exception: The suffix -in is still found in oxidoreductase and the reaction
the names of some digestive enzymes, catalyzed is shown below
E.g., trypsin, chymotrypsin, and pepsin
Transferase
2. Type of reaction catalyzed by an enzyme is often
used as a prefix • A transferase is an enzyme that catalyzes the
– E.g., Oxidase - catalyzes an oxidation transfer of a functional group from one molecule
reaction, to another
– E.g., Hydrolase - catalyzes a hydrolysis • Two major subtypes:
reaction – Transaminases - catalyze transfer of an
3. Identity of substrate is often used in addition to amino group to a substrate
the type of reaction – Kinases - catalyze transfer of a phosphate
– E.g. Glucose oxidase, pyruvate group from adenosine triphosphate (atp)
carboxylase, and succinate to a substrate
dehydrogenase
Lyase
• Predict the function of the following enzymes.
a. Maltase • A lyase is an enzyme that catalyzes the addition
b. Lactate dehydrogenase of a group to a double bond or the removal of a
c. Fructose oxidase group to form a double bond in a manner that does
d. Maleate isomerase not involve hydrolysis or oxidation
– Dehydratase: effects the removal of the
Answers: components of water from a double bond
a. Hydrolysis of maltose – Hydratase: effects the addition of the
b. Removal of hydrogen from lactate ion components of water to a double bonds
c. Oxidation of Fructose
d. Rearrangement (isomerization) of maleate ion Isomerase, and ligase

• An isomerase is an enzyme that catalyzes the

Six Major Classes isomerization (rearrangement of atoms)
- Enzymes are grouped into six major classes based reactions.
on the types of reactions they catalyze • A ligase is an enzyme that catalyzes the formation
of a bond between two molecules involving ATP
hydrolysis:
– ATP hydrolysis is required because such
reactions are energetically unfavorable
– Require the simultaneous input of energy
obtained by a hydrolysis of ATP to ADP

Oxidoreductase
• An oxidoreductase enzyme catalyzes an
oxidation–reduction reaction:
– Oxidation and reduction reactions are
always linked to one another
 • Induced Fit Model:
Answers; A. Transferase, B. Lyase – Substrate contact with enzyme will
change the shape of the active site
Models of enzyme action – Allows small change in space to
accommodate substrate (e.g., how a hand
Enzyme Active Site fits into a glove)

• The active site: Relatively small part of an Forces that determine substrate binding
enzyme’s structure that is actually involved in
catalysis: • H-bonding
– Place where substrate binds to enzyme • Hydrophobic interactions
– Formed due to folding and bending of the • Electrostatic interactions
protein.
– Usually a “crevice like” location in the Enzyme specificity
enzyme
– Some enzymes have more than one • Absolute Specificity:
active site – An enzyme will catalyze a particular
reaction for only one substrate
– This is most restrictive of all specificities
(not common)
– E.g., Urease is an enzyme with absolute
specificity
• Stereochemical Specificity:
– An enzyme can distinguish between
stereoisomers.
– Chirality is inherent in an active site
(amino acids are chiral compounds)
– L-Amino-acid oxidase - catalyzes
reactions of L-amino acids but not of D-
amino acids.
• Group Specificity:
– Involves structurally similar compounds
that have the same functional groups.
– E.g., Carboxypeptidase: Cleaves amino
acids one at a time from the carboxyl end
of the peptide chain
Enzyme Substrate Complex
• Linkage Specificity:
– Involves a particular type of bond
• Needed for the activity of enzyme
irrespective of the structural features in
• Intermediate reaction species formed when
the vicinity of the bond
substrate binds with the active site
– Considered most general of enzyme
• Orientation and proximity is favorable and
specificities
reaction is fast
– E.g., Phosphatases: Hydrolyze
phosphate–ester bonds in all types of
phosphate esters
TWO MODELS FOR SUBSTRATE BINDING TO
ENZYME
Factors that affect enzyme activity
• Lock-and-Key model:
Temperature
– Enzyme has a pre-determined shape for
• Higher temperature results in higher kinetic
the active site
energy which causes an increase in number of
– Only substrate of specific shape can bind
with active site
 reactant collisions, therefore there is higher
activity.
• Optimum temperature: Temperature at which the
rate of enzyme catalyzed reaction is maximum
• Optimum temperature for human enzymes is 37ºc
(body temperature)
• Increased temperature (high fever) leads to
decreased enzyme activity

Enzyme concentration

• Enzymes are not consumed in the reactions they

catalyze
• At a constant substrate concentration, enzyme
activity increases with increase in enzyme
concentration
– The greater the enzyme concentration,
the greater the reaction rate.

Ph

• Ph changes affect enzyme activity

• Drastic changes in ph can result in denaturation of
proteins
• Optimum ph: ph at which enzyme has maximum
activity
• Most enzymes have optimal activity in the ph range
of 7.0 - 7.5
• Exception: Digestive enzymes
• Pepsin: Optimum ph = 2.0
• Trypsin: Optimum ph = 8.0 • Describe the effect that each of the following
changes would have on the rate of a reaction that
SUBSTRATE CONCENTRATION involves the substrate sucrose and the intestinal
enzyme sucrase.
• Substrate Concentration: At a constant enzyme a. Decreasing the sucrase concentration
concentration, the enzyme activity increases with b. Increasing the sucrose concentration
increased substrate concentration. c. Lowering the temperature to 10ºc
• Substrate saturation: the concentration at which it Raising the ph from 6.0 to 8.0 when the optimum
reaches its maximum rate and all of the active ph is 6.2
sites are full
• Turnover Number: Number of substrate Answers:
molecules converted to product per second per A. Decrease rate
enzyme molecule under conditions of optimum B. Increase rate
temperature and Ph C. Decrease rate
D. Decrease rate
Enzyme inhibition Regulation of enzyme activity

• Enzyme Inhibitor: a substance that slows down or • Cellular processes continually produces large
stops the normal catalytic function of an enzyme amounts of an enzyme and plentiful amounts of
by binding to it. products if the processes are not regulated.
• Competitive Inhibitors: Compete with the • General mechanisms involved in regulation:
substrate for the same active site – Proteolytic enzymes and
– Will have similar charge & shape zymogenscovalent modification of
– Noncompetitive Inhibitors: Do not enzymes
compete with the substrate for the same – Feedback control Regulation of enzyme
active site activity by various substances produced
– Binds to the enzyme at a location other within a cell
than active site – The enzymes regulated are allosteric
enzymes
Reversible competitive inhibition
Properties of allosteric enzymes
• A competitive enzyme inhibitor: resembles an
enzyme substrate in shape and charge • All allosteric enzymes have a quarternary
• Binds reversibly to an enzyme active site and the structure:
inhibitor remains unchanged (no reaction occurs) – Composed of two or more protein chains
• The enzyme - inhibitor complex formation is via • Have at least two of binding sites:
weak interactions (hydrogen bonds, etc.). – Substrate and regulator binding site
• Competitive inhibition can be reduced by simply • Active and regulatory binding sites are distinct
increasing the concentration of the substrate. from each other:
– Located independent of each other
Reversible noncompetitive inhibition – Shapes of the sites (electronic geometry)
are different
• A noncompetitive enzyme inhibitor decreases • Binding of molecules at the regulatory site causes
enzyme activity by binding to a site on an enzyme changes in the overall three dimensional structure
other than the active site. of the enzyme:
• Causes a change in the structure of the enzyme – Change in three dimensional structure of
and prevents enzyme activity. the enzyme leads to change in enzyme
• Increasing the concentration of substrate does not activity
completely overcome inhibition. – Some regulators increase enzyme
• Examples: Heavy metal ions Pb2+, Ag+, and Hg2+. activity – activators
– Some regulators decrease enzyme
Irreversible activity - inhibitors

• An irreversible enzyme inhibitor inactivates Feedback control

enzymes by forming a strong covalent bond with
the enzyme’s active site. • Feedback Control: A process in which activation
– The structure is not similar to enzyme’s normal or inhibition of the first reaction in a reaction
substrate sequence is controlled by a product of the
– The inhibitor bonds strongly and increasing reaction sequence.
substrate concentration does not reverse the • Regulators of a particular allosteric enzyme may
inhibition process be:
– Enzyme is permanently inactivated. – Products of entirely different pathways of
– E.g., Chemical warfare agents (nerve reaction within the cell
gases) and organophosphate insecticides – Compounds produced outside the cell
(hormones)
Proteolytic enzymes and zygomens Antibiotics that inhibit enzyme activity

• 2nd mechanism of regulating enzyme activity: Sulfa Drug

– Production of enzymes in an inactive
forms (zymogens) • Many derivatives of sulfanilamide collectively
– Zymogens are “turned on” at the called sulfa drugs exhibit antibiotic activities
appropriate time and place • Sulfanilamide is structurally similar to PABA (p-
– Example: proteolytic enzymes: Most aminobenzoic acid)
digestive and blood-clotting enzymes are • Many bacteria need PABA to produce coenzyme,
proteolytic enzymes folic acid
– Hydrolyze peptide bonds in proteins • Sulfanilamide is a competitive inhibitor of
– Proteolytic enzymes are generated in an enzymes responsible for converting PABA to
inactive form and then converted to their folic acid in bacteria
active form • Folic acid deficiency retards bacterial growth and
that eventually kills them
• Sulfa drugs don’t affect humans because we
absorb folic acid from our diet

Penicillins

• Accidently discovered by Alexander Fleming in

1928
• Several naturally occurring penicillins and numerous
synthetic derivatives have been produced
• All have structures containing a four-membered
Beta-lactam ring fused with a five-membered
thiazolidine ring
Covalent modification of enzymes • Selectively inhibits transpeptidase by covalent
modification of serine residue
• 3rd Mechanism for regulation of enzyme activity • Transpeptidase catalyzes the formation of peptide
• Covalent modification: A process in which cross links between polysaccharides strands in
enzyme activity is altered by covalently bacterial cell walls
modifying the structure of the enzyme:
– Involves adding or removing a group Cipro
from an enzyme
• Most common covalent modification: addition • The antibiotic ciprofloxacin hydrochloride (Cipro for
and removal of phosphate group: short)
– Phosphate group is often derived from an • Considered the best broad-spectrum antibiotics
ATP molecule. because it is effective against skin and bone
– Addition of the phosphate infections as well as against infections involving the
(phosphorylation) catalyzed by a Kinase urinary, gastrointestinal, and respiratory systems
enzyme • It is the drug of choice for treatment of traveler’s
– Removal of the phosphate group diarrhea
(dephosphorylation) catalyzed by a • Bacteria are slow to acquire resistance to Cipro.
phosphatase enzyme. – Biochemical threats associated with
– Phosphate group is added to (or removed terrorism has thrust Cipro into the spotlight
from) the R group of a serine, tyrosine, or because it is effective against anthrax.
threonine amino acid residue in the
enzyme regulated.
Medical Uses of Enzyme Vitamin B

• Diagnose certain diseases: • The preferred and alternative names for the B
– Enzymes produced in certain vitamins
organ/tissues if found in blood may • Thiamin (vitamin B1)
indicate certain damage to that • Riboflavin (vitamin B2)
organ/tissue • Niacin (nicotinic acid, nicotinamide, vitamin B3)
• Vitamin B6 (pyridoxine, pyridoxal,
General Characteristics of Vitamins pyridoxamine)
• Folate (folic acid)
• Organic compounds • Vitamin B12 (cobalamin)
• Must be obtained from dietary sources • Pantothenic acid (vitamin B5)
• Human body can’t synthesize in enough amounts • Biotin
• Essential for proper functioning of the body • Exhibit structural diversity
• Needed in micro and milligram quantities Major function: B Vitamins are components of
• 1 Gram of vitamin B is sufficient for 500,000 coenzymes
people
• Enough vitamin can be obtained from balanced Fat Soluble Vitamins
diet
• Supplemental vitamins may be needed after Vitamins A, D, E, K
illness
• Many enzymes contain vitamins as part of their • Involved in plasma membrane processes
structures - conjugated enzymes • More hydrocarbon like with fewer functional
• Two Classes groups
– Water Soluble and Fat Soluable • Vitamin A
• Synthetic and natural vitamins are same – Has role in vision - only 1/1000 of
– 13 Known vitamins\ vitamin A is in retina
– 3 Forms of vitamin A are active in the
Water soluble Vitamins body
– Derived from b-carotine
Vitamin C
• Humans, monkeys, apes and guinea pigs need Functions of Vitamin A
dietary vitamins
• Co-substrate in the formation of structural protein • Vision: In the eye- vitamin A combines with opsin
collagen protein to form the visual pigment rhodopsin
• Involved in metabolism of certain amino acids which further converts light energy into nerve
• 100 mg/day saturates all body tissues - Excess impulses that are sent to the brain.
vitamin is excreted • Regulating Cell Differentiation - process in which
• RDA (mg/day): immature cells change to specialized cells with
– Great Britain: 30 function.
– United States and Canada: 60 – Examples: Differentiation of bone
– Germany: 75 marrow cells white blood cells and red
blood cells.
• Maintenance of the healthy of epithelial tissues
via epithelial tissue differentiation.
– Lack of vitamin A causes such surfaces to
become drier and harder than normal.
• Reproduction and Growth: In men, vitamin A
participates in sperm development. In women,
normal fetal development during pregnancy
requires vitamin A.
Functions of Vitamin D – a —OH group present on carbon 2’ in
ribose
• Two forms active in the body: Vitamin D2 and D3 – a —H atom in 2-deoxyribose
• Sunshine Vitamin: Synthesized by UV light from • RNA and DNA differ in the identity of the sugar
sun unit in their nucleotides.
• It controls correct ratio of Ca and P for bone
mineralization (hardening) Nitrogen-Containing Heterocyclic Bases
• As a hormone it promotes Ca and P absorption in • There are a total five bases (four of them in most
intestine of DNA and RNAs)
• Three pyrimidine derivatives - thymine (T),
Functions of Vitamin E cytosine (C), and uracil (U)
• Two purine derivatives - adenine (A) and guanine
• Four forms of Vitamin Es: a-, b-, g- and d-Vitamin (G)
E • Adenine (A), guanine (G), and cytosine (C) are
• Alpha-tocopherol is the most active biological found in both DNA and RNA.
active form of Vitamin E • Uracil (U): found only in RNA
• Peanut oils, green and leafy vegetables and whole Thymine (T) found only in DNA.
grain products are the sources of vitamin E
• Primary function: Antioxidant – protects against Phosphate
oxidation of other compounds • Phosphate - third component of a nucleotide, is
derived from phosphoric acid (H3PO4)
Functions of Vitamin K • Under cellular pH conditions, the phosphoric acid
is fully dissociated to give a hydrogen phosphate
• Two major forms; K1 and K2 ion (HPO42-)
• K1 found in dark green, leafy vegetables
• K2 is synthesized by bacteria that grow in colon Nucleotide Formation
• Dietary need supply: ~1/2 synthesized by bacteria • The formation of a nucleotide from sugar, base,
and 1/2 obtained from diet and phosphate is visualized below.
• Active in the formation of proteins involved in – Phosphate attached to C-5’ and base is
regulating blood clotting attached to C-1’ position of pentose

NUCLEIC ACID

Nucleotides: Building Blocks of Nucleic Acids

• Nucleic Acids: Polymers in which repeating unit
is nucleotide
• A Nucleotide has three components:
– Pentose Sugar: Monosaccharide
– Phosphate Group (PO43-)
– Heterocyclic Base Nucleotide Nomenclature

Base

Phosphate Sugar

Pentose Sugar
• Ribose is present in RNA and 2-deoxyribose is
present in DNA
• Structural difference:
Primary Nucleic Acid Structure
• Sugar-phosphate groups are referred to as nucleic
acid backbone - Found in all nucleic acids
• Sugars are different in DNA and RNA

The DNA Double Helix

• Nucleic acids have secondary and tertiary

structure
• The secondary structure involves two
polynucleotide chains coiled around each other in
a helical fashion
• The poly nucleotides run anti-parallel (opposite
Primary Structure
directions) to each other, i.e., 5’ - 3’ and 3’ - 5’
• A ribonucleic acid (RNA) is a nucleotide polymer in
• The bases are located at the center and hydrogen
which each of the monomers contains ribose, a
bonded (A=T and GΞC)
phosphate group, and one of the heterocyclic bases
• Base composition: %A = %T and %C = %G)
adenine, cytosine, guanine, or uracil
– Example: Human DNA contains 30%
• A deoxyribonucleic acid (DNA) is a nucleotide
adenine, 30% thymine, 20% guanine and
polymer in which each of the monomers contains
20% cytocine
deoxyribose, a phosphate group, and one of the
heterocyclic bases adenine, cytosine, guanine, or
• DNA Sequence: the sequence of bases on one
thymine.
polynucleotide is complementary to the other
polynucleotide
• Structure: Sequence of nucleotides in DNA or RNA
• Complementary bases are pairs of bases in a
• Primary structure is due to changes in the bases
nucleic acid structure that can hydrogen-bond to
• Phosphodiester bond at 3’ and 5’ position
each other.
• 5’ end has free phosphate and 3’ end has a free OH
• Complementary DNA strands are strands of DNA
group
in a double helix with base pairing such that each
• Sequence of bases read from 5’ to 3’
base is located opposite its complementary base.
• Example :
• List of bases in sequential order in the direction
from the 5’ end to 3’ end of the segment:
• 5’-A-A-G-C-T-A-G-C-T-T-A-C-T-3’
• Complementary strand of this sequence will be:
3’-T-T-C-G-A-T-C-G-A-A-T-G-A-5’

Base Pairing
• One small and one large base can fit inside the
DNA strands:
– Hydrogen bonding is stronger with A-T
and G-C
– A-T and G-C are called complementary
bases
Comparison of the General Primary Structures of Nucleic
Acids and Proteins Practice Exercise

• Backbone: -Phosphate-Sugar- Nucleic acids Predict the sequence of bases in the DNA strand
• Backbone: -Peptide bonds – Proteins complementary to the single DNA strand shown below:
 5’ A–A–T–G–C–A–G–C–T 3’ Overview of Protein Synthesis

Answer: • Protein synthesis is directly under the direction of

3’ T–T–A–C–G–T–C–G–A 5’ DNA
• Proteins are responsible for the formation of skin,
Replication of DNA Molecules hair, enzymes, hormones, and so on
• Replication: Process by which DNA molecules • Protein synthesis can be divided into two phases.
produce exact duplicates of themselves – Transcription – A process by which DNA
• Old strands act as templates for the synthesis of directs the synthesis of mRNA molecules
new strands – Translation – a process in which mRNA
• DNA polymerase checks the correct base pairing isdeciphered to synthesize a protein
and catalyzes the formation of phosphodiester molecule
linkages
• The newly synthesized DNA has one new DNA
strand and old DNA strand
• DNA polymerase enzyme can only function in the
5’-to-3’ direction Ribonucleic Acid; Differences between RNA and DNA
• Therefore one strand (top; leading strand ) grows molecules
continuously in the direction of unwinding
• The lagging strand grows in segments (Okazaki • The sugar unit in the backbone of RNA is ribose;
fragments) in the opposite direction it is deoxyribose in DNA.
• The segments are latter connected by DNA ligase • The base thymine found in DNA is replaced by
• DNA replication usually occurs at multiple sites uracil in RNA
within a molecule (origin of replication) • RNA is a single-stranded molecule; DNA is
• DNA replication is bidirectional from these sites double-stranded (double helix)
(replication forks) • RNA molecules are much smaller than DNA
• Multiple-site replication enables rapid DNA molecules, ranging from 75 nucleotides to a few
synthesis thousand nucleotides

Chromosomes Types of RNA Molecules

• Upon DNA replication the large DNA molecules
interacts with histone proteins to fold long DNA • Heterogeneous nuclear RNA (hnRNA): Formed
molecules. directly by DNA transcription.
• The histone–DNA complexes are called • Post-transcription processing converts the
chromosomes: hnRNA to mRNA
– A chromosome is about 15% by mass • Messenger RNA: Carries instructions for protein
DNA and 85% by mass protein. synthesis (genetic information) from DNA
– Cells of different kinds of organisms – The molecular mass of mRNA varies
have different numbers of chromosomes. with the length of the protein
– Example: Number of chromosomes in a • Small nuclear RNA: Facilitates the conversion of
human cell 46, a mosquito 6, a frog 26, a hnRNA to mRNA.
dog 78, and a turkey 82 – Contains from 100 to 200 nucleotides
• Chromosomes occur in matched (homologous) • Ribosomal RNA (rRNA): Combines with specific
pairs. proteins to form ribosomes - the physical site for
– Example: The 46 chromosomes of a protein synthesis. Ribosomes have molecular
human cell constitute 23 homologous masses on the order of 3 million
pairs
• Transfer RNA (tRNA): Delivers amino acids to
the sites for protein synthesis
- tRNAs are the smallest (75–90 nucleotide
units)
 – Exon - a gene segment that codes for
genetic information
– Intron – a DNA segments that interrupt a
genetic message
• The splicing process is driven by
snRNA
• Alternative splicing - A process by which several
different protein variants are produced from a
single gene
– The process involves excision of one or
Transcription: RNA Synthesis
more exons
Transcription
Transcriptome
• Transcription: A process by which DNA directs
• Transcriptome: All of the mRNA molecules that
the synthesis of mRNA molecules
can be generated from the genetic material in a
– Two-step process - (1) synthesis of
genome.
hnRNA and (2) editing to yield mRNA
– Transcriptome is different from a genome
molecule
– Responsible for the biochemical
• Gene: A segment of a DNA base sequence
complexity created by splice variants
responsible for the production of a specific
obtained by hnRNA.
hnRNA/mRNA molecule
– Most human genes are ~1000–3500
nucleotide units long
– Genome: All of the genetic material (the
total DNA) contained in the
chromosomes of an organism
– Human genome is about 20,000–25,000
genes

Steps in the Transcription Process:

The Genetic Code
• Unwinding of DNA double helix to expose some
bases (a gene): • The base sequence in a mRNA determines the
– The unwinding process is governed by amino acid sequence for the protein synthesized.
RNA polymerase • The base sequence of an mRNA molecule
• Alignment of free ribonucleotides along the involves only 4 different bases - A, C, G, and U
exposed DNA strand (template) forming new • Codon: A three-nucleotide sequence in an mRNA
base pairs molecule that codes for a specifi c amino acid
• RNA polymerase catalyzes the linkage of – Based on all possible combination of
ribonucleotides one by one to form mRNA bases A, G, C, U” there are 64 possible
molecule codes
• Transcription ends when the RNA polymerase • Genetic code: The assignment of the 64 mRNA
enzyme encounters a stop signal on the DNA codons to specific amino acids (or stop signals)
template: – 3 of the 64 codons are termination codons
– The newly formed RNA molecule and (“stop” signals)
the RNA polymerase enzyme are
released Characteristics of Genetic Code

Post-Transcription Processing: Formation of mRNA • The genetic code is highly degenerate:

– Many amino acids are designated by
• Involves conversion of hnRNA to mRNA more than one codon.
• Splicing: Excision of introns and joining of exons
 – Arg, Leu, and Ser - represented by six • The loop opposite to the open end of tRNA is the
codons. site for a sequence of three bases called an
– Most other amino acids - represented by anticodon.
two codons • Anticodon - a three-nucleotide sequence on a
– Met and Trp - have only a single codon. tRNA molecule that is complementary to a codon
– Codons that specify the same amino acid on an mRNA molecule.
are called synonyms
• There is a pattern to the arrangement of synonyms Translation: Protein Synthesis
in the genetic code table.
– All synonyms for an amino acid fall • Translation – a process in which mRNA codons
within a single box in unless there are are deciphered to synthesize a protein molecule
more than four synonyms • Ribosome – an rRNA–protein complex - serves
– The significance of the “single box” as the site of protein synthesis:
pattern - the first two bases are the same – Contains 4 rRNA molecules and ~80
– For example, the four synonyms for proteins - packed into two rRNA-protein
Proline - CCU, CCC, CCA, and CCG. subunits (one small and one large)
– ~65% rRNA and 35% protein by mass
• The genetic code is almost universal: – A ribosome’s active site – Large subunit
– With minor exceptions the code is the – Ribosome is a RNA catalyst
same in all organisms – The mRNA binds to the small subunit of
– The same codon specifies the same the ribosome.
amino acid whether the cell is a bacterial
cell, a corn plant cell, or a human cell. Five Steps of Translation Process
• An initiation codon exists:
– The existence of “stop” codons (UAG, • Activation of tRNA: addition of specific amino
UAA, and UGA) suggests the existence acids to the 3’-OH group of tRNA.
of “start” codons. • Initiation of protein synthesis: Begins with
– The codon - coding for the amino acid binding of mRNA to small ribosomal subunit
methionine (AUG) functions as initiation such that its first codon (initiating codon AUG)
codon. occupies a site called the P site (peptidyl site)
• Elongation: Adjacent to the P site in an mRNA–
PRACTICE EXERCISE: ribosome complex is A site (aminoacyl site) and
the next tRNA with the appropriate anticodon
binds to it.
• Termination: The polypeptide continues to grow
via translocation until all necessary amino acids
are in place and bonded to each other.
• Post-translational processing – gives the protein
Answers: the final form it needs to be fully functional
a. 3’ GCG–GCA–UCA–ACC–GGG–CCU–CCU 5’
b. 3’ GCG–ACC–CCU–CCU 5’ Efficiency of mRNA Utilization

Anticodons and tRNA Molecules • Polysome (polyribosome): complex of mRNA

• During protein synthesis amino acids do not and several ribosomes
directly interact with the codons of an mRNA • Many ribosomes can move simultaneously along
molecule. a single mRNA molecule
• tRNA molecules as intermediaries deliver amino • The multiple use of mRNA molecules reduces the
acids to mRNA. amount of resources and energy that the cell
• Two important features of the tRNA structure expends to synthesize needed protein.
• The 3’ end of tRNA is where an amino acid is • In the process – several ribosomes bind to a single
covalently bonded to the tRNA. mRNA - polysomes.
 – Many human diseases are of viral origin,
e. g. Common cold, smallpox, rabies,
influenza, hepatitis, and AIDS
• Viruses attach to the host cell on the outside cell
surface and proteins of virus envelope catalyze
the breakdown of the cell membrane and forms a
hole
• Viruses then inject their DNA or RNA into the
host cell
Mutation • The viral genome is replicated, proteins coding
• An error in base sequence reproduced during for the viral envelope are produced in hundreds of
DNA replication copies.
• Errors in genetic information is passed on during • Hundreds of new viruses are produced using the
transcription. host cell replicated genome and proteins in short
• The altered information can cause changes in time
amino acid sequence during protein synthesis and
thereby alter protein function
• Such changes have a profound effect on an Vaccines
organism. • Inactive virus or bacterial envelope
• Antibodies produced against inactive viral or
Mutagens bacterial envelopes will kill the active bacteria
and viruses
• Mutations are caused by mutagens
• A mutagen is a substance or agent that causes a Recombinant DNA and Genetic Engineering
change in the structure of a gene:
– Radiation and chemical agents are two • DNA molecules that have been synthesized by
important types of mutagens splicing a sequence of segment DNA (usually a
– Ultraviolet, X-ray, radioactivity and gene) from one organism to the DNA of another
cosmic radiation are mutagenic –cause organism
cancers • Genetic Engineering (Biotechnology):
– Chemical agents can also have mutagenic – The study of biochemical techniques
effects that allow the transfer of a “foreign”
• E.g., HNO2 can convert cytosine gene to a host organism and produce the
to uracil protein associated with the added gene
• Nitrites, nitrates, and – Bacterial strains such as E. coli inserted
nitrosamines – can form nitrous with circular plasmids, and/or yeast cells
acid in cells carrying vectors containing foreign
• Under normal conditions mutations are repaired genes are used for this purpose
by repair enzymes – Plasmids (double stranded DNA)
replicate independently in bacteria or
Nucleic Acids and Viruses yeast

Viruses Recombinant DNA Production Using Bacterial Plasmid

• Viruses: Tiny disease causing agents with outer
protein envelope and inner nucleic acid core • Dissolution of cells:
• They can not reproduce outside their host cells – E. coli cells of a specific strain
(living organisms) containing the plasmid of interest are
• Invade their host cells to reproduce and in the treated with chemicals to dissolve their
process disrupt the normal cell’s operation membranes and release the cellular
• Virus invade bacteria, plants animals, and contents
humans: • Isolation of plasmid fraction:
 – The cellular contents are fractionated to DNA Sequencing
obtain plasmids
• Cleavage of plasmid DNA: • DNA sequencing is a method by which the base
– Restriction enzymes are used to cleave sequence in a DNA molecule (or a portion of it)
the double-stranded DNA is determined.
• Gene removal from another organism: • Discovered in 1977 by Fredrick Sanger
– Using the same restriction enzyme the • Concept in DNA sequencing:
gene of interest is removed from a • Selective interruption of polynucleotide synthesis
chromosome of another organism using 2’,3’-dideoxyribonucleotide triphosphates
• Gene–plasmid splicing: (ddNTPs).
– The gene (from Step 4) and the opened
plasmid (from Step 3) are mixed in the
presence of the enzyme DNA ligase to
splice them together.
• Uptake of recombinant DNA:
The recombinant DNA prepared in stept 5 are transferred
to a live E. coli culture where they can be replicated,
trasncribed and translated
• This interruption of synthesis leads to the
• Transformed cell can reproduce a large number of formation of every possible nucleotide site
identical cells –clones: mixture.
– Clones are the cells that have descended • These nucleotides are labeled using radioactive
from a single cell and have identical dNTP during their synthesis.
DNA • The radiolablled nucleotides are then separated
• Given bacteria grow very fast, within few hours on a gel by electrophoresis
1000s of clones will be produced
• Each clone can synthesize the protein directed by Basic steps involved in DNA sequencing
foreign gene it carries • Step 1: Cleavage of DNA using restriction
enzymes: Restriction enzymes are used to cleave
The Polymerase Chain Reaction the large DNA molecule into smaller fragments
(100–200 base pairs).
• The polymerase chain reaction (PCR) is a method • Step 2: Separation into individual components:
for rapidly producing multiple copies of a DNA The mixture of small DNA fragments generated
nucleotide sequence (gene). by the restriction enzymes is separated into
• This method allows to produce billions of copies individual components via gel electrophoresis
of a specific gene in a few hours. techniques.
• PCR is very easy to carryout and the requirements • Step 3: Separation into single strands: A given
are: DNA fragment is separated into its two strands by
– Source of gene to be copied chemical methods to use it as a template in step
– Thermostabel DNA polymerase 4.
– Deoxynucleotide triphosphates (dATP,
dGTP, dCTP and dTTP)
– A set of two oligonucleotides with
complementary sequence to the gene
(primers)
– Thermostable plastic container and
– Source of heat