PROTEIN

Proteins are large biomolecules, or

macromolecules, consisting of one or more
long chains of amino acid residues.
Proteins are polymers of amino acids linked together
by what is called “ Peptide bond”
A linear chain of amino acid residues is called
a polypeptide. A protein contains at least one
long polypeptide.
PROTIENS ARE LARGE MOLECULES
CONSISITING OF AMINO ACIDS LINKED BY
PEPTIDE BONDS.
(Peptide bond is formed when carboxyl group
of one amino acids reacts with an amino
group of the other amino acids.)
 Amino acids
Amino acids are biologically important organic compounds
containing amine (-NH2) and carboxylic(-COOH) acid
functional groups.
The key elements of an amino acid are carbon, hydrogen,
oxygen, and nitrogen, though other elements are found in the
side-chains of certain amino acids.
About 500 amino acids are known and can be classified in
many ways.
They can be classified according to the core structural
functional groups' locations as alpha- (α-), beta- (β-), gamma-
(γ-) or delta- (δ-) amino acids;
other categories relate to polarity, pH level, and side-chain
group type (aliphatic, acyclic, aromatic, containing hydroxyl or
sulfur, etc.).
Structure of amino acids:
Each amino acid has 4 different groups attached to α-
carbon ( which is C-atom next to COOH).
These 4 groups are : amino group, COOH group,
Hydrogen atom and side Chain (R)
• At physiological PH (7.4), -COOH gp is dissociated forming a
negatively charged carboxylate ion (COO-) and amino gp is
protonated forming positively charged ion (NH3+) forming
Zwitter ion.
 Classification of amino acids

 I- Chemical classification: According to number of COOH and

NH2 groups i.e. according to net charge on amino acid.
A- Monobasic, monocarboxylic amino acids i.e. neutral or
uncharged:
• Amino acids that have an equal amount of carboxylic acids and
amino groups.
• a - Valine
• b- Leucine
• c- Serine and d- Threonine
B- Basic amino acids: Contain two or more NH2
groups or nitrogen atoms that act as base i.e.
can bind proton.
At physiological pH, basic amino acids will be
positively charged.
e.g. a- Lysine b- Arginine c-
Histidine

C- Acidic Amino acids: at physiological pH will carry

negative charge.
e.g. Aspartic acid (aspartate) and Glutamic acid
(glutamate). see structures in hand out.
II- Classification according to polarity of side chain (R):
 A- Polar amino acids: in which the functional group attached to the
α-carbon (R in RCH(NH3+)COO−) has hydrophilic properties.
OR
in which R contains polar hydrophilic group so can forms hydrogen
bond with H2O. In those amino acids, R may contain:
1- OH(hydroxyl) group : as in serine, threonine and tyrosine
2- SH(sulfur) group : as in cysteine
3- amide group: as in glutamine and aspargine
4- NH2 group or nitrogen act as a base (basic amino acids ): as lysine,
arginine and histidine
5- COOH group ( acidic amino acids): as aspartic and glutamic .
B- Non polar amino acids: R is hydrophobic group
which can’t enter in hydrogen bond formation.
They however possess groups such as hydroxyl,
sulfhydyl and amid and participte in hydrogen bonding
of protein structure.
9 amino acids are non polar (glycine, alanine, valine,
leucine, isoleucine, phenyl alanine, tryptophan, proline
and methionine).
They have no charge on the R group
 III- Nutritional classification:
 1- Essential amino acids: These amino acids can’t be
formed/ synthesized by the body and so, it is essential to be
taken in diet. Their deficiency affects growth, health and
protein synthesis.
M= methionine, T= tryptophan,
Th= threonine, P= phenyl alanine
 2- Semiessential / conditionally essential amino acids:
These are formed in the body but not in sufficient amount for
body requirements.
 In certain conditions like surgery, wound, sepsis, thermal
injury,etc. the non essential amino acids become
conditionally essential. Need to be supplemented in the diet.
Arginine and Histidine are semiessential
3- Non essential amino acids: These are the rest of amino
acids that are formed in the body in amount enough for
adults and children. They are the remaining 10 amino acids.
IV- Metabolic classification:
according to metabolic or degradation products of amino acids
they may be:
1- Ketogenic amino acids: which give ketone bodies,i.e fat
can be synthesized from these amino acid. Lysine and
Leucine are the only pure ketogenic amino acids.
2- Mixed ketogenic and glucogenic amino acids: which
give both ketonbodies and glucose. These are: isoleucine,
phenyl alanine, tyrosine and tryptophan.
3- Glucogenic amino acids: Which synthesize glucose.
These amino acids by catabolism yields products that enter in
glycogen and glucose formation.
Amphoteric properties of amino acids: that is they have
both basic and acidic groups and so can act as base or acid.
Neutral amino acids (monobasic, monocarboxylic) exist in
aqueous solution as “ Zwitter ion” i.e. contain both positive
and negative charge. Zwitter ion is electrically neutral and
can’t migrate into electric field.
 Isoelectric point (IEP) = is the pH at which the zwitter ion
is formed. e.g IEP of alanine is 6.
 Chemical properties of amino acids:
1- Reactions due to COOH group:
- Salt formation with alkalis, ester formation with alcohols,
amide formation with amines and decarboxylation
2- Reactions due to NH2 group: deamination and reaction
with ninhydrin reagent.
- Ninhydrin reagent reacts with amino group of amino acid
yielding blue colored product. The intensity of blue color
indicates quantity of amino acids present.
3- Reactions due to side chain (R):

1- Millon reaction: for tyrosine gives red colored mass

2- Rosenheim reaction: for trptophan and gives violet
ring.
3- Pauly reaction: for imidazole ring of histidine: gives
yellow to reddish product
4- Sakagushi test: for guanido group of arginine andgives
red color.
5- Lead sulfide test (sulfur test): for sulfur containing
amino acids as cysteine give brown color.
 Peptides and Proteins
20 amino acids are commonly found in protein.
These 20 amino acids are linked together through “peptide
bond forming peptides and proteins (what’s the difference?).
 - The chains containing less than 50 amino acids are called
“peptides”, while those containing greater than 50 amino
acids are called “proteins”.
Peptide bond formation:
α-carboxyl group of one amino acid (with side chain R1)
forms a covalent peptide bond with α-amino group of
another amino acid ( with the side chain R2) by removal of
a molecule of water. The result is : Dipeptide ( i.e. Two
amino acids linked by one peptide bond).
By the same way, the dipeptide can then forms a second
peptide bond with a third amino acid (with side chain R3) to
give Tripeptide.
Repetition of this process generates a polypeptide or protein
of specific amino acid sequence.
 Peptide bond formation:

- Each polypeptide chain starts on the left side by free amino group of
the first amino acid enter in chain formation . It is termed (N- terminus).
- Each polypeptide chain ends on the right side by free COOH group of
the last amino acid and termed (C-terminus).
 High orders of Protein structure

• A functional protein is not just a polypeptide chain, but

one or more polypeptides precisely twisted, folded and
coiled into a molecule of unique shape (conformation).
This conformation is essential for some protein function e.g.
Enables a protein to recognize and bind specifically to
another molecule e.g. hormone/receptor; enzyme/substrate
and antibody/antigen.
Protein structure:
There are four levels of protein structure (primary,
secondary, tertiary and quaternary)
Primary structure:
• The primary structure of a protein is its unique
sequence of amino acids.
– Lysozyme, an enzyme that attacks bacteria,
consists of a polypeptide chain of 129 amino
acids.
– The precise primary structure of a protein is
determined by inherited genetic information.
– At one end is an amino acid with a free amino
group the (the N-terminus) and at the other is
an amino acid with a free carboxyl group the
(the C-terminus).
2- Secondary structure:
•After synthesis, polypeptide chains are folded or pleated
into different shapes, called their Secondary Structure.
•Secondary structure is held together by many Hydrogen
bonds, overall giving the shape great stability.
•[Secondary Structure: The way in which the primary
structure of a polypeptide chain folds.]
•Hydrogen bond formation takes place between hydrogen
of –NH group of peptide bond and the carbonyl oxygen of
another peptide bond. According to H-bonding there are
two main forms of secondary structure:
Intrachain hydrogen bonding
β-sheets: Is another
form of secondary
structure in which two
or more polypeptides
(or segments of the
same peptide chain) are
linked together by
hydrogen bond between
H- of NH- of one chain
and carbonyl oxygen of
adjacent chain (or
segment).
• Interchain hydrogen
bonding
Super secondary structure or Motifs :
occurs by combining secondary
structure.
The combination may be:
α-helix- turn- α-helix- turn…..etc
Or:
β-sheet -turn- β-sheet-turn………etc
Or:
α-helix- turn- β-sheet-turn- α-helix
3. Tertiary structure – secondary
structure may be further folded,
superfolded, twisted about itself
forming various configuration.
• Is determined by a variety of
interactions (bond formation) among
R groups and between R groups and
the polypeptide backbone.
a. The weak interactions include:
 Hydrogen bonds among polar
side chains
 Ionic bonds between charged R
groups (basic and acidic amino
acids)
 Hydrophobic interactions
among hydrophobic ( non polar)
R groups.
b. Strong covalent bonds include disulfide bridges,
that form between the sulfhydryl groups (SH) of
cysteine monomers, stabilize the structure.

4. Quaternary structure: results from the combination

of two or more polypeptide subunits held together by
non-covalent interaction like H-bonds, ionic or
hydrophobic interactions.
• Examples on protein having quaternary structure:
- Collagen is a fibrous protein of three polypeptides
(trimeric) that are supercoiled like a rope.
- This provides the structural strength for their role in
connective tissue.
- Hemoglobin is a globular protein with four polypeptide
chains (tetrameric)
- Insulin : two polypeptide chains (dimeric)
 Classification of proteins
On the bases of size and shape.
On the bases of functional properties.
On the bases of solubility and physical
properties.
I. On the bases of size and shape.
[Link] proteins
• the axial ratio of length/breadth is more than
10
• They have primarily mechanical and
structural functions, providing support to
the cells as well as the whole [Link]
appear hair like with long thin fiber.
2. Globular proteins
 Axial ratio of length and breadth is less than 10
 Most of the proteins belong to this class.
 They have a compact and more or less spherical
structure, more complex than fibrous proteins.
 motifs, domains, tertiary and quaternary structures
are found, in addition to the secondary structures.
 They are generally soluble in water but can also be
found inserted into biological membranes
(transmembrane proteins), thus in a hydrophobic
environment.
 Unlike fibrous proteins, that have structural and
mechanical functions, they act as:
 Enzymes ,hormones; membrane transporters and
receptors; transporters of triglycerides, fatty acids
and oxygen in the blood; immunoglobulins or
antibodies; grain and legume storage proteins.
 Functional classification proteins

Structural proteins- keratin of hair and nails,

collagen of bones.
Enzymes or catalytic proteins- hexokinase,
pepsin.
Transport proteins- hemoglibin, serum albumin.
Hormonal proteins- growth hormones.
Contractile proteins- actin, myosisn.
Storage proteins- ovalbumin, glutelin.
Genetic proteins- nucleoproteins.
Défense proteins- snake venoms,
immunoglobulins.
On the bases of solubility and physical properties.
I- Simple proteins: i.e. on hydrolysis gives only amino
acids. Also known as homoproteins, they are made up of
only amino acids.
1- Albumins:
• Soluble in water and in dilute salt solutions.
• They are coagulable by heat.
• Ex – animal albumins
• Ovalbumin (egg)
• Lactalbumin(milk)
• plant albumins –
• ligumelin(legumes)
• Leucosin (cereals)
2. Globulins
• Insoluble in water but soluble in dilute neutral salt
solutions.
• They are coagulable by heat.
• Globulins bind with heme(hemopexin),
metals( transferrin), and
carbohydrates(immunoglobulins).
• Example – ovaglobulin (egg), lactaglobulin(milk),
legumin(legumes).
3. Gliadins
• Insoluble in water, dilute salt solutions and absolute
alcohol, but soluble in 50-80% ethanol.
• Ex – gliadin(wheat), hordein(barly).
[Link]
• Are small molecules soluble in water,dilute ammonia, dilute acids
and alkalies.
• They are non coagulable by heat.
• They form nucleoproteins with nuclic acids.
• Ex- Salmine, sardinine and cyprinin.
[Link]
• Basic proteins rich in arginine and histidine.
• soluble in water, dilute salt solutions and dilute acids.
• Insoluble in ammonia.
• They are not readily coagulate on heating.
• They form conjugated proteins with DNA.
6-Scleroproteins:
• These are fibrous protein having very low solubility.
• They have great stability and form supporting
structures of animal.
• They are structural proteins, not digested .
• include: keratin, collagen and elastin.
 II. Conjugated proteins
On hydrolysis, give protein part and non protein part.
Sometimes also called heteroproteins, and subclassified
into:
1- Phosphoproteins: These are proteins conjugated with
phosphate group. Phosphorus is attached to OH group of
serine or threonine.
e.g. Casein of milk and vitellin of yolk.
2- Lipoproteins: These are proteins conjugated with lipids.
 Functions:
a- help lipids to transport in blood
b- Enter in cell membrane structure helping lipid soluble
substances to pass through cell membranes.
3- Glycoproteins:
proteins conjugated with sugar (carbohydrate)
e.g. – Mucin, immunoglobulins.
- Some hormones such as erythropoeitin
- present in cell membrane structure
- blood groups.

4- Nucleoproteins: These are basic proteins ( e.g. histones)

conjugated with nucleic acid (DNA or RNA).
e.g. Nucleohistone and nucleoprotamine
a- chromosomes: have proteins conjugated with DNA
b- Ribosomes: have proteins conjugated with RNA
5- Metalloproteins: These are proteins conjugated with metal
like iron, copper, zinc, ……
a- Iron-containing proteins: Iron may present in heme such
as in
- hemoglobin (Hb)
- myoglobin ( protein of skeletal muscles and cardiacmuscle)
- cytochromes,
- catalase, peroxidases (destroy H2O2)
- tryptophan pyrrolase (desrtroy indole ring of tryptophan).
- Ex- ferritin (Fe)
b- Copper containing proteins:
e.g. - Ceruloplasmin
- Oxidase enzymes such as cytochrome oxidase.
c- Zn containing proteins: e.g. Insulin and carbonic
anhydrase ex- Carbonicanhydrase(Zn)
d- Mg containing proteins: e.g. Kinases and
phosphatases.

6-Chromoproteins:These are proteins conjugated with

pigment. e.g.
- All proteins containing heme (Hb, myoglobin)
- Melanoprotein: e.g proteins of hair which contain
melanin.
 III. Derived proteins
 Produced by various physical and chemical factors from simple and
conjugated proteins. Theses are the denatured or degraded products of
simple and conjugated proteins.
 Divided into two major groups.
1 Primery derived proteins
 This group includes denatured and coagulated proteins obtained from the
native proteins by heat, X-rays, UV-rays, acid, alkali, vigorous shaking
etc.
 They have same molecular weight but differ in solubility, precipitation
and crystallization.
 There is only intramolecular rearrangement in which peptide bonds
remain intact.
i. Proteans-
insoluble products formed from certain globulins by the action of water,
very dilute acids and enzymes
Ex- Myosan(from myosin), Elastan(from elastin), Fibrin(from Fibinogen)
ii. Metaproteins
Further action of acids and alkalies on proteins produces
metaproteins which are generally soluble in dilute acids and
alkalies but insoluble in neutral solvents.
Ex- acid and alkali metaproteins
iii. Coagulated proteins.
They are insoluble products formed by the action of heat or
alcohol on native proteins.
Ex- cooked egg albumin, cooked meat.
2. secondary derived proteins
They are formed by progressive hydrolysis of native protein
at their peptide linkage.
According to their relative average molecular size, they are
roughly called proteoses, peptones and peptides.
i. Proteoses- these hydrolytic products of proteins soluble in
water and coagulated by heat
ii. Peptones- these are hydrolytic products of proteoses.
Water soluble
Non-coagulable
Obtained by enzymatic digestion of protein
iii. Peptides-
• they have very small number of amino acids
linked together by peptide bonds, and named
accordingly – Dipeptide, Tripeptid etc
• These are water soluble
• Non- coagulable
Protein synthesis is simply the "making of proteins."
biosynthesis is the process whereby biological cells
generate new proteins
the process by which amino acids are linearly arranged
into proteins through the involvement of ribosomal RNA,
transfer RNA, messenger RNA, and various enzymes.
In order to make even one protein, the body must seek the
aid of messenger RNA, transfer RNA, DNA, amino acids,
ribosomes, and multiple enzymes.
 Vocabulary of Terms
Aminoacyl-tRNA: tRNA with an amino acid
attached
Peptidyl-tRNA: tRNA with peptide attached
Nascent chain: peptide chain in act of being
assembled
P-site: Site on ribosome where peptidyl-tRNA
sits
A-site: Site on ribosome where incoming
aminoacyl-tRNA binds
Peptidyl transferase: Enzyme that forms peptide
bond
GENES are long strands of DNA on chromosomes
Transcription
First Step: Copying of genetic information from DNA
to RNA called Transcription
Why? transcription ??/?
DNA has the genetic code for the protein that
needs to be made, but proteins are made by the
ribosomes—ribosomes are outside the nucleus in
the cytoplasm.
DNA is too large to leave the nucleus (double
stranded), but RNA can leave the nucleus (single
stranded).
Part of DNA temporarily unzips and is used as a
template to assemble complementary nucleotides
into messenger RNA (mRNA).
mRNA then goes through the pores of the nucleus
with the DNA code and attaches to the ribosome.
Translation
Decoding of mRNA into a protein is called Translation.
Transfer RNA (tRNA) carries amino acids from the
cytoplasm to the ribosome.
These amino acids come from the food we eat. Proteins
we eat are broken down into individual amino acids and
then simply rearranged into new proteins according to
the needs and directions of our DNA.
A series of three adjacent bases in an mRNA molecule
codes for a specific amino acid—called a codon.
A triplet of nucleotides in tRNA that is complementary
to the codon in mRNA—called an anticodon.
Each tRNA codes for a different amino acid.
mRNA carrying the DNA instructions and tRNA carrying
amino acids meet in the ribosomes.
Amino acids are joined together to make a protein.
Role of nuclic acid
Messenger RNA (mRNA) is the blueprint for
construction of a protein. Has the message
(codon) responsible for directing the
sequence of amino acids in the protein
Ribosomal RNA (rRNA) is part of robosomes
which are factories manufacturing proteins
in the cell. It is the construction site where
the protein is made.
Transfer RNA (tRNA) is the truck delivering
the proper amino acid to the site at the
right time.

mRNA
mRNA Binding
Binding

Initiation Complex
Initiation Complex
f-Met-tRNA Met
f-Met-tRNAffMet
Initiation
Initiation factors
factors

Elongation
Elongation
Elongation
Elongation factors
factors

Termination
Termination
Termination
Termination factors
factors
Translation is the process of converting the
mRNA codon sequences into an amino acid
sequence.
The initiator codon (AUG) codes for the amino
acid N-formylmethionine (f-Met).
No transcription occurs without the AUG codon. f-
Met is always the first amino acid in a
polypeptide chain, although frequently it is
removed after translation. The intitator
tRNA/mRNA/small ribosomal unit is called the
initiation complex. The larger subunit attaches to
the initiation complex. After the initiation phase
the message gets longer during the elongation
phase.
Preparation of protein synthesis
 Amino acids must be activated before they are used for protein synthesis
 It is the step in which each of participating amino acid reacts with ATP
to form amino acid AMP complex and pyrophosphate
 Each different tRNA molecule has a unique shape and chemical
composition that is recognised by a specific tRNA-activating enzyme
 The enzyme (aminoacyl-tRNA synthetase) first binds the amino acid to
a molecule of ATP (to form an amino acid-AMP complex.
 The amino acid is then transferred to the 3'-end of the appropriate tRNA,
attaching to a terminal CCA sequence on the acceptor stem and releasing
the AMP molecule
 The tRNA molecule with an amino acid attached is thus said to be
'charged' and is now capable of participating in translation
 The energy in the bond linking the tRNA molecule to the amino acid
will be used in translation to form a peptide bond between adjacent
amino acids.
 Initiation
 Protein synthesis is initiated by binding of small unit
30s of ribosome on 5’ side of mRNA.
 This process is assisted by three initiation factors
IF1,IF2 and IF3.
 The bound 30s ribosome move on the mRNA till it
enconter the initiation codon-5’ AUG.
 The ribosome has three sites A (amino acyl)site,
P(pepttidyl) site, E (exit ) site.
 Peptide synthesis always starts from methionine
(Met). Therefore, the initial aminoacyl-tRNA is Met-
tRNAiMet, where the subscript "i" specifies "initiation".
 In bacteria, the methionine of the initial aminoacyl-
tRNA has been modified by the addition of a formyl
group (HCO) to its amino group. The modified
methionine is called formylmethionine (fMet),
At this step the 30S subunit of ribosome is
joined to 50S subunit and initiation prossess is
complete.
Elongation step
 Three elongation factors (EF Tu, EF Ts, EF G) assist the
elongation of the polypeptide.
 (EF refers to elongation factor and TU/Ts refers to temperature
unstable/stable)
 The charged tRNA molecule carrying amino acid, enters the
ribosome at A site.
 The anticodon present on the anticodon arm of tRNA locates and
binds to the complementary codon of mRNA at A site.
 A peptide bond is formed between two amino acid, one present on
the tRNA at P site and another newly arrived at A site.
 The process is catalysed by the enzyme peptidyl transferase
located on ribosome.
 The ribosome moves one codon along the mRNA in the 3’
direction.
The complex then shifts along the mRNA to
the next triplet, opening the A site. With
this , tRNA dipeptide complex at A site is
pulled to P site. This process is called
translocation.
The new tRNA enters at the A site.
 Termination
Protein synthesis will terminate when the ribosome
arrives at one of three stop codons UAA, UAG and
UGA.
The termination process is assisted by special
proteins called termination factors(release factors,
RF1,RF2 and RF3) which recognize the stop codons.
Their association stimulates the release of the
peptidyl-tRNA from the ribosome.
Subsequently, the released peptidyl-tRNA divides
into tRNA and a newly synthesized peptide chain.
The ribosome also divides into the large and small
subunits, ready for synthesizing another peptide.
PROTEIN TARGETING
Protein targeting or protein sorting is
the biological mechanism by which proteins
are transported to the appropriate
destinations in the cell or outside of it after
synthesis.
Proteins can be targeted to the inner space
of an organelle, different intracellular
membranes, plasma membrane, or to
exterior of the cell via secretion.
This delivery process is carried out based on
information contained in the protein itself.
Some cytoplasmic proteins are targeted to a
particular site in the cell because they
contain a specific peptide sequence that
 Targeting signals

Targeting signals are the pieces of information that

enable the cellular transport machinery to correctly
position a protein inside or outside the cell.
This information is contained in the polypeptide chain or
in the folded protein.
The continuous stretch of amino acid residues in the
chain that enables targeting are called signal peptides or
targeting peptides.
Can be located on N-, C-terminus or in the middle of the
protein
Protein targeting - Protein has to be correctly localized
to perform proper function.
Receptors – plasma membrane
DNA polymerase – nucleus
Catalase – peroxisomes
All proteins begin to be synthesized on
cytosolic ribosomes. Sorting or
translocation can occur
1. Co-translational translocation
(translocation during the process of
translation), and
2. Post-translational translocation
(translocation after the process of
translation is complete).
If the protein is for cytosolic functions, the
synthesis will be finished on free ribosomes and
the peptide is released into the cytosol.

If the protein is destined for nucleus,

mitochondria or peroxisomes the synthesis is
also finished on cytoplasmic ribosomes and the
peptide is released to the cytosol (to be sorted
later or post-translationally).

If the protein is going to be secreted from the cell

or it destined for the membranes the ribosome
with the nascent peptide is targeted to the ER
(ER becomes rough) and sorting is done during
translation (co- translationally).
Co-translational translocation
Most proteins that are secretory ( membrane-bound, or reside
in the endoplasmic reticulum (ER), golgi or endosomes use
the co-translational translocation pathway.
This process begins with the N-terminal signal peptide of the
protein being recognized by a signal recognition
particle(SRP) while the protein is still being synthesized on
the ribosome.
The synthesis pauses while the ribosome-protein complex is
transferred to an SRP receptor(signal receptor particle) on the
ER in eukaryotes, and the plasma membrane in prokaryotes.
There, the nascent protein is inserted into the translocon, a
membrane-bound protein conducting channel composed of
the Sec61 translocation complex in eukaryotes, and the
homologous SecYEG complex in prokaryotes.
Post-translational translocation
Even though most secretory proteins are co-translationally
translocated, some are translated in the cytosol and later
transported to the ER/plasma membrane by a post-
translational system.
In prokaryotes this requires certain cofactors such as SecA
and SecB.
This pathway is poorly understood in eukaryotes, but is
facilitated by Sec62 and Sec63, two membrane-bound
proteins.
In addition, proteins targeted to other destinations, such as
mitochondria, chloroplasts, or peroxisomes, use specialized
post-translational pathways. Also, proteins targeted for the
nucleus are translocated post-translation. They pass through
the nuclear envelope via nuclear pores.
Transport of the new protein into mitochondria
Most mitochondrial proteins are encoded by nuclear DNA
Only very few are encoded by mitochondrial DNA and
synthesized on mitochondrial ribosomes
 Mitochondrial targeting signals
Usually located at N-terminus of precursor polypeptide
Usually removed in mitochondrial matrix
 Receptor/translocation channels in mitochondria
Tom – translocase of the outer mitochondrial membrane
Tim - translocase of the inner mitochondrial membrane
Mitochondrial proteins are synthesized in cytosol as
precursors.
 Bind to cytosolic chaperones(Hsp 70) to keep them unfolded
until they ready to be translocated .
Uses Energy from ATP
Some outer membrane proteins insert
themselves in the membrane.
Intermembrane space proteins remain there
and fold.
Protein destined to matrix passes through
Tom 40 and then Tim (inner membrane
translocon)
Transport into peroxisomes
Proteins are synthesized and fully folded in cytosol
Fully functional, fully folded protein is transported!
Import requires ATP hydrolysis
Peroxisome targeting sequences
PTS1 on C-terminus
PTS2 on N-terminus
Peroxins - peroxisome transport receptors. Bind to proteins
with PTS1 and move to the translocation channel
The complex is transported through the membrane
Protein is released
Peroxin is recycled
Transport into the nucleus
All proteins found in the nucleus are synthesized in the
cytoplasm
Examples:
Histones
Ribosomal proteins
DNA and RNA polymerases
Transcription factors
Transport requires nuclear localization sequences (NLS)
Transport occurs through the nuclear pores.
Nuclear import receptor (Importin α and β)
Energy from GTP
Fully folded proteins are transported
Co-translational translocation
across the ER membrane
ER signal sequence emerges
The binding by a signal-recongition particles
(SRP).
SRP delivers the ribosome/nascent polypeptide
complex to the SRP receptor in the ER
membrane, and GTP binding
synthesized on the rough ER
Diseases due to defective protein targeting
1. ZELLWEGER SYNDROME
2. PRIMARY HYPEROXALURIA
3. FAMILIAL HYPERCHOLESTROLEMIA
4. CYSTIC FIBROSIS
5. INCLUSION CELL DISEASE
Glycosylayion
 The protein can me modified by attachment of sugar residue to the
protein by an enzymatic process. This process is known as
glycosylation.
 Modification of protein can take place along with its synthesis(co-
translation) or after protein synthesis(post translation modification).
 A large number of membrane proteins which are usually trans-
membrane proteins are glycosylated.
 The carbohydates which are usually added include glucose, galactose,
mannose, xylose, fructose, etc
 The carbohydrate chains attached to the target proteins serves various
functions.
 The co-translational glycosylation may help in correct protein folding.
 Polysaccharides linked at the amide nitrogen of aspargine in the
proteim confer stability on some secreted glycoprotein.
 Glycosylayion may also play a role in cell-cell adhesion (a mechanism
employed by cells of immune system.)
Chromatography
Chromatography is the collective term for a set of
laboratory techniques for the separation of mixtures.
The mixture is dissolved in a fluid called the mobile
phase, which carries it through a structure holding
another material called the stationary phase.
The various constituents of the mixture travel at
different speeds, causing them to separate.
There are two main categories of
chromatography: preparative and analytical.
1. Analytical work (which may be used in an
environmental lab to look for pollutants) uses
small sample sizes; the objective is to separate
compounds in order to identify them.
2. Preparative work (which may be used in
the pharmaceutical industry) uses large
quantities of samples and collects the output in
bulk; the point of the chromatography here is
to remove impurities from a commercial
product.
Basics of Chromatography
In any chromatographic technique, a stationary
phase usually a solid, thick liquid, or bonded coating that
stays fixed in one place, and a mobile phase or
eluent (usually a liquid or gas) moves through it or across it.
A sample to be separated, when placed on the stationary
phase, will gradually move along in the same direction as the
mobile phase.
If a sample compound (or analyte) has no interaction with
the stationary phase, it will run right through and come out of
the system (elute) at the same rate as the mobile phase.
If an analyte has no interaction with the mobile phase, it will
stick directly to the stationary phase and never elute. Neither
of these are good outcomes.
In a well-designed chromatography process, the chemist will
choose stationary and mobile phases that will both have at
least some interaction with the analytes.
Any individual sample molecule will interact first with one
phase and then the other, but the fraction of each analyte
overall in each phase will remain constant.
This distribution ratio among the selected phases must
differ for each analyte in order for them to separate.
(Compounds will not separate chromatographically if they
have the same distribution ratio on a particular system.)
Separation is only the first part of chromatography. In
Tswett's original experiment, he could see where the various
compounds were by color.
Most chemicals are colorless, however, so there must be
a detector to notice when compounds elute.
While there are various types of detectors, their output is
usually a chart with peaks corresponding to the various
compounds (a chromatogram).
The amount of time any given analyte stays on the column
is its retention time and the area under its peak is
proportional to its concentration, so the chemist can figure
out how much of one analyte is present relative to the other
compounds.
Rf = distance travelled by the compound from origin
distance travelled by the solvent from origin
One thing chromatography alone cannot do is identify the
molecular structure of the sample compounds; it can
separate them from each other and detect them, but it does
not tell what they are. Other kinds of analytical work would
be needed for identification.
Types of Chromatography
There are hundreds of stationary phase, mobile phase,
and detector combinations in wide use throughout the
chemical, medical, and biotechnology industries, but we
can identify few types.
1. Size-exclusion chromatography (SEC)
Is a chromatographic method in which molecules in
solution are separated by their size, and in some
cases molecular weight.
It is usually applied to large molecules or
macromolecular complexes such as proteins and
industrial polymers.
Techniqe is based upon the use of porous gel in the form
of insoluble beads placed in the column.
As a solution of protein passed through the column, small
proteins can penetrate into the pores of the bead and,
therefore, are retarded in their rate of travel through the
column.
In larger proteins, a protein is the less likely to enter the
pores.
Different beads with different pore size can be used
depending on the desired protein size separation profile.
Advantages
The advantages of this method include good separation of
large molecules from the small molecules with a minimal
volume of eluate,
Various solutions can be applied without interfering with the
filtration process.
The technique is generally combined with others that further
separate molecules by other characteristics, such as acidity,
basicity, charge, and affinity for certain compounds.
With size exclusion chromatography, there are short and
well-defined separation times and narrow bands, which lead
to good sensitivity.
Ion Exchange Chromatography
The most popular method for the purification of proteins
and other charged molecules is ion exchange
chromatography.
In cation exchange chromatography positively charged
molecules are attracted to a negatively charged solid
support.
Conversely, in anion exchange chromatography, negatively
charged molecules are attracted to a positively charged
solid support.
Each protein exhibits a distinct overall net pH. some
proteins will be negatively charged some will be positively
charged at the same pH. This property of protein is the
basis for ion exchange chromatography.
Fine cellulose resins are used that are either negatively
charged(cation exchanger) or positively charged (anion
exchanger).
Proteins of opposite charge to resin are retained as the
solution of protein passed through the column.
The bound proteins are then eluted out by passing a solution
of ion bearing a charge opposite to that of the column. By
utilizing a gradient of increasing ionic strength, proteins
with increasing affinity for the resin are progressively eluted
Affinity chromatography
Affinity chromatography is a method of separating
biochemical mixtures based on a highly specific interaction
such as that between antigen and antibody, enzyme and
substrate, or receptor and ligand.
Uses
Affinity chromatography can be used to:
Purify and concentrate a substance from a mixture into a
buffering solution
Reduce the amount of a substance in a mixture
Discern what biological compounds bind to a particular
substance
Purify and concentrate an enzyme solution.
A column of beads bearing high affinity
compound can be prepared and solution of
protein passed through the column.
The protein will bind and unbound molecule
will elute out.
Then bound protein is separated/ eluted by
passing a solution of unbound soluble high
affinity compound through the column
Electrophoresis
is a method for separation and analysis of
macromolecules (DNA, RNA and proteins) and
their fragments, based on their size and charge.
It is separation technique in which the
components are separated due to their varying
behaviour under the influence of applied electric
field.
It is defined as the migration of charged
molecules under the influence of external
electric field.
The major requirement of the component to be
subjected to electrophoresis is that the
component should be charged molecule.
It is mostly used in separation of
biological substances like
Proteins
Nucleic acids
Peptides
Polysaccharides
Amino acids
Oligosaccharides
Nucleosides
Organic acids
Small cations and anions in the body
Nucleic acid molecules are separated by
applying an electric field to move the
negatively charged molecules through a
matrix of agarose or other substances.
Shorter molecules move faster and migrate
farther than longer ones because shorter
molecules migrate more easily through the
pores of the gel. This phenomenon is called
sieving.
Proteins are separated by charge in agarose
because the pores of the gel are too large to
sieve proteins.
Gel electrophoresis can also be used for
separation of nanoparticles.
(Low level of electroendosmosis)
Buffers –
Most common buffer used is TRIS
(Tris (hydroxymethyl)
aminomethane or THAM)
 The equipment and supplies necessary for conducting agarose
gel electrophoresis are relatively simple and include:
1. An electrophoresis chamber and power supply
2. Gel casting trays, which are available in a variety of sizes and
composed of UV transparent plastic. The open ends of the trays
are closed with tape while the gel is being cast, then removed
prior to electrophoresis.
3. Sample combs, around which molten medium is poured to
form sample wells in the gel.
4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or
Tris-borate-EDTA (TBE).
5. Loading buffer, which contains something dense (e.g.
glycerol) to allow the sample to “fall” into the sample wells,
and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has
proceeded.
6. Staining: DNA molecules are easily visualized under
an ultraviolet. Alternatively, nucleic acids can be stained
after electrophoretic separation by soaking the gel in a
solution of ethidium bromide. When intercalated into
double stranded DNA, fluorescence of this molecule
increases greatly.
7. Transilluminator (an ultraviolet light box), which is
used to visualize stained DNA in gels.
1. To prepare gel, agarose powder is mixed with electrophoresis
buffer to the desired concentration, and heated in a microwave
oven to melt it.
2. Ethidium bromide is added to the gel (final concentration 0.5
ug/ml) to facilitate visualization of DNA after electrophoresis.
3. After cooling the solution to about 60oC, it is poured into a
casting tray containing a sample comb and allowed to solidify
at room temperature.
4. After the gel has solidified, the comb is removed, taking care
not to rip the bottom of the wells.
5. The gel, still in plastic tray, is inserted horizontally into the
electrophoresis chamber and is covered with buffer.
6. Samples containing DNA mixed with loading buffer are then
pipetted into the sample wells, the lid and power leads are
placed on the apparatus, and a current is applied.
7. The current flow can be confirmed by observing bubbles
coming off the electrodes.
8. DNA will migrate towards the positive electrode, which is
usually colored red, in view of its negative charge.
9. The distance DNA has migrated in the gel can be judged
by visually monitoring migration of the tracking dyes like
bromophenol blue and xylene cyanol dyes.
 Protein sequencing is a technique to determine the
amino acid sequence of a protein, as well as which conformation the
protein adopts and the extent to which it is complexed with any non-
peptide molecules.
 A biological sequence is a single, continuous molecule of nucleic
acid or protein. It can be thought of as a multiple inheritance class
hierarchy. One hierarchy is that of the underlying molecule type:
DNA, RNA, or protein.
Protein sequencer
 A protein sequencer is a machine that is used to determine the
sequence of amino acids in a protein.
 They work by tagging and removing one amino acid at a time,
which is analysed and identified. This is done repetitively for the
whole polypeptide, until the whole sequence is established.
 This method has generally been replaced by nucleic acid technology,
and it is often easier to identify the sequence of a protein by looking
at the DNA that codes for it.
nitric
(Tpp- thiamine pyrophosphate)
Nitrogen assimilation
is the formation of organic nitrogen
compounds like amino acids from inorganic
nitrogen compounds present in the
environment.
Plants absorb nitrogen from the soil in the
form of nitrate (NO3−) and ammonium
(NH4+).
Organisms like plants, fungi and certain
bacteria that cannot fix nitrogen gas (N2)
depend on the ability to assimilate nitrate
or ammonia for their needs.
Other organisms, like animals, depend
entirely on organic nitrogen from their food.
Ammonium ions are absorbed by the plant via
ammonia transporters.
Nitrate is taken up by several nitrate
transporters that use a proton gradient to power
the transport.
Nitrogen is transported from the root to the
shoot via the xylem in the form of nitrate,
dissolved ammonia and amino acids.
Usually(but not always) most of the nitrate
reduction is carried out in the shoots while the
roots reduce only a small fraction of the
absorbed nitrate to ammonia.
Ammonia (both absorbed and synthesized) is
incorporated into amino acids via the glutamine
synthetase-glutamate synthase (GS-GOGAT)
pathway.
 Urea Cycle

Conversion of NH3 to urea for

excretion
Most terrestrial animals convert excess nitrogen
to urea, prior to excreting it.
Urea is less toxic than ammonia.
The Urea Cycle occurs mainly in liver. First 2
reactions in mitochondria and rest in cytosol.
The 2 nitrogen atoms of urea enter the Urea
Cycle as NH3 (produced mainly via Glutamate
Dehydrogenase) and as the amino N of
aspartate.
The NH3 and HCO3- (carbonyl C) that will be part
of urea are incorporated first into carbamoyl
phosphate.
Sources of Ammonia:
1. Ammonia is produced in the body from
the different tissues by Amino acid
catabolism.
2. Purine and Pyramidine catabolism.
3. The other source is from the dietary
proteins and from urea present in fluids.
Glutamine Synthetase GLUTAMINE is the
important plasma transport form of
nitrogen from muscle.
Enzymes involved in mitochondria
[Link] phosphate synthase I
[Link] transcarbamylase
Enzymes involved in cytosol
1. Arginino succintate synthase
2. Arginino succinase
3. Arginase
1. Transport of excess ammonia by glutamine:
Excess ammonia is toxic to animal tissues.
Glutamine synthetase catalyses the
synthesis of glutamine by adding the
ammonia to glutamate at the expense of ATP
hydrolysis.
Glutamine is a non-toxic carrier of ammonia.
It is transported to liver or kidney via blood.
In the liver & kidney, glutamine is
reconverted to glutamate and ammonia by
glutaminase.
Ammonia is incorporated in urea cycle to
form urea and then it will be excreted
through kidneys.
Step:2 (synthesis of Citrulline)
Ornithine transcarbamoylase – mitochondrial
Catalyses addition (condensetion ) of
ornithine to the carbonyl group of Carbamoyl
phosphate. To give citrulline
Step:3 (synthesis of Argininosuccinate)
Catalysed by cytosolic Argininosuccinate
synthase, citrulline and aspartate are
condatnsed to form Argininosuccinate–
cytosolic ATP --AMP
Step:4 (Cleavage of Argininosuccinate)
Argininosuccinase /Argininosuccinate lyase
cytosolic enzyme Argininosuccinate --
Arginine + Fumarate
Step:5 ( Cleavage of Arginine to Ornithine and
Urea) by Enzyme: Arginase – cytosolic
In final step of urea cycle Arginase cleave urea
from arginine, regenerating cytosolic ornithine,
which can be transported to mytochondrial
matrix for another round of urea cycle
ENERGETICS
2 ATPs are utilized for the synthesis of
carbamoyl phosphate.
1 ATP is converted to AMP and
Ppi(pyrophosphate) to produce
Arginosuccinate
NH4+ + CO2 + aspartate + 3 ATP --->urea +
fumarate + 2 ADP + AMP + 4 Pi
Hemes and Chlorophylls
Definition -Heme or haem is a cofactor consisting
of an Fe2+ (ferrous) ion contained in the centre of a
large heterocyclic organic ring called a porphyrin,
made up of four pyrrolic groups joined together by
methine bridges.
Not all porphyrins contain iron, but a substantial
fraction of porphyrin-containing metalloproteins
have heme as their prosthetic group; these are
known as hemoproteins.
Hemes are most commonly recognized as
components of hemoglobin, the red pigment in
blood
Also found in a number of other biologically
important hemoproteins such as myoglobin,
cytochrome, catalase, heme peroxidase,
and endothelial nitric oxide synthase.
Functions
Hemoproteins have diverse biological functions
including the transportation of diatomic gases,
chemical catalysis, diatomic gas detection, and
electron transfer.
The heme ion serves as a source or sink of electrons
during electron transfer or redox chemistry.
In peroxidase reactions, the porphyrin molecule also
serves as an electron source.
In the transportation or detection of diatomic gases,
the gas binds to the heme ion.
During the detection of diatomic gases, the binding
of the gas ligand to the heme ion induces
conformational changes in the surrounding protein.
Major hemes
There are several biologically important kinds of
heme:
The most common type is heme B;
other important types include heme A and
heme C.
heme O
(note-Isolated hemes are commonly designated by
capital letters while hemes bound to proteins are
designated by lower case letters.)
Heme l is the derivative of heme B which is
covalently attached to the protein of
lactoperoxidase, eosinophil peroxidase, and
thyroid peroxidase.
Heme l is one important characteristic of animal
Heme m is the derivative of heme B
covalently bound at the active site of
myeloperoxidase.
Heme D is another derivative of heme B,
Heme D is the site for oxygen reduction to
water of many types of bacteria at low
oxygen tension.
Heme S is related to heme B
Heme S is found in the hemoglobin of marine
worms.
Structure of Heme B
 ENZYMES

Are biological molecules(proteins)

which can accelerate/increase the
rate of reaction
Enzymes are biological molecules (proteins) that act as
catalysts and help complex reactions occur everywhere in life.
Let's say you ate a piece of meat. Proteases would go to work
and help break down the peptide bonds between the amino
acids.
Enzymes are macromolecular biological catalysts.
Enzymes accelerate, or catalyze, chemical reactions.
The molecules at the beginning of the process are called
substrates and the enzyme converts these into different
molecules, called products.
Almost all metabolic processes in the cell need enzymes in
order to occur at rates fast enough to sustain life.
The study of enzymes is called enzymology. Enzymes are
known to catalyze more than 5,000 biochemical reaction types.
Most enzymes are proteins,
Who discovered the first enzyme?
In 1833, French chemist Anselme Payen discovered the
first enzyme, diastase.
How do you name an enzyme?
An enzyme's name is often derived from its substrate or
the chemical reaction it catalyzes, with the word ending
in -ase.
Examples are lactase, alcohol dehydrogenase and DNA
polymerase.
Different enzymes that catalyze the same chemical
reaction are called isozymes.
Properties
 Are protein in nature
 All enzymes are proteins but all proteins are not enzymes
 Accelerate the rate of the reaction , they are biocatalyst
 They highly specific in their function
 They are very fast in their action
 Little amount of enzyme is required to carry out biochemical
reactions.
 Active site – is a site on enzyme to which substrate binds, the
arrangement of atoms in an active site is well defined, this results in
specificity of enzyme.
 chemical environment inside the active site
permits a chemical reaction to proceed more easily.
 Enzymes are active at specific pH and temperature.
 The other chemical groups required for enzyems i.e co-factor and co-
enzymes.
 Cofactors, are inorganic molecules, mostly metal ions. Mg2+, Fe 2+, Zn +2
 Coenzymes are non-protein organic molecules that are mostly derivatives of
vitamins soluble in water by phosphorylation; they bind apoenzyme to proteins
to produce an active holoenzyme. NADH, FADH, biotin, Folic acid.
 Holoenzymes- a complete catalytically active enzymes
togather with its coenzymes or metal ions is called
holoenzymes.
 Cofactors with an apoenzyme are called a holoenzyme, which
is the active form.
 Holoenzymes = Apoenzyme (protein part) + prosthetic
group (cofactor/ coenzyme)
 Apoenzyme- the protein part of the enzyme on its own
without its cofactor is called apoenzyme
 Enzymes without their necessary cofactors are called
apoenzymes, which are the inactive form of an enzyme.
 Prosthetic group- a metal ion or cofactor/ coenzyme that is
covalently attached to the apoenzyme is called prosthetic
 Enzymes are highly specific in action.
 Enzymes remain chemically unchanged at the end
of the reaction.
 Enzymes are required in minute amounts.
Naming
An enzyme's name is often derived from its
substrate or the chemical reaction it catalyzes,
with the word ending in -ase.
Examples are lactase, alcohol dehydrogenase
and DNA polymerase.
Different enzymes that catalyze the same
chemical reaction are called isozymes.
The
International Union of Biochemistry and Molecul
ar Biology
have developed a nomenclature for enzymes,
the EC numbers; each enzyme is described by a
sequence of four numbers preceded by "EC".
The first number broadly classifies the enzyme
based on its mechanism.
The top-level classification is:
EC 1. Oxidoreductases: catalyze oxidation/reduction
reactions, transfer of H and O atoms or electrons from one
substance to another
AH + B  A + BH(Reduced)
A+O AO (oxidized)
a. oxidases- in this case oxygen is act electron acceptor
b. peroxidases- the reaction in which hydrogen atom is
removed from substrate and added to water molecule.
H +H2O  H202(hydrogen peroxide)
c. Dehydrogenases- re the enzymes removing the
hydrogen atom from the substrate. H2O2 is not formed. ex-
lactate dehydrogenase
d. oxygenases- one or more than one
oxygen molecules are added to substrate.
ex- cytochromes
EC 2. Transferases: transfer a functional group
/specific group from one substrate to another
subsrate. (e.g. a methyl or phosphate group)
AB + C  A + BC
Ex- transaminases , hexokinses, thexokinases
a. Transaminases
b. Transketolases
c. Transaldolases
d. Transcarboxylases(co2)
e. Transphosporylases(phosphate)
f. Transglycolases –(carbohydrate)
EC 3. Hydrolases: catalyze/involved in the hydrolysis
of various bonds( hydrolysis is addition of H 2O)
Formation of two products from substrate by
hydrolysis.
AB+ H2O  AOH + BH
Ex- lipase, choline, esterase, pepsin, trypsin, urease,
a. Peptidases- break the peptide bond by hydrolysis
b. Esterases- act on ester bond
c. Glyosylases- break down glycogenic bonds

EC 4. Lyases: cleave various bonds by means other

than hydrolysis and oxidation
ex- aldolase, tumaras, histidase, decarboxylase
Enzymes which catalysis the removal or addition to the
substare other than oxygen, hydrogen and water
molecule.
C-C , C-N, or C-S bonds may be cleaved.
EC 5. Isomerases: catalyze isomerization
changes within a single molecule.
Intermolecul rearrangement, i.e is isomerization
changes within a single molecule
AB  BA
Ex- isomerase, mutase, racemase, epimerases

EC 6. Ligases: join two molecules with covalent

[Link] known as synthatase
Join together two molecules by synthesis of new
C-O, C-S, C-N or C-C bond with simultaneous
break down of ATP,(require ATP)
X + Y + ATP  XY + ADP + Pi
Ex- glutamine synthetase, succinate thiokinase.
Control of activity
There are five main ways that enzyme activity is controlled in the cell.
1. Regulation
 Enzymes can be either activated or inhibited by other molecules.
 For example, the end product(s) of a metabolic pathway are often
inhibitors for one of the first enzymes of the pathway (usually the first
irreversible step, called committed step), thus regulating the amount
of end product made by the pathways.
 Such a regulatory mechanism is called a
negative feedback mechanism, because the amount of the end product
produced is regulated by its own concentration.
 Negative feedback mechanism can effectively adjust the rate of
synthesis of intermediate metabolites according to the demands of the
cells.
 It prevents the excess manufacture of end products. The control of
enzymatic action helps to maintain a stable internal environment in
living organisms
2. Post-translational modification
Examples of post-translational modification include
phosphorylation and glycosylation.
For example, in the response to insulin, the phosphorylation
of multiple enzymes, including glycogen synthase, helps
control the synthesis or degradation of glycogen and allows
the cell to respond to changes in blood sugar.
Another example of post-translational modification is the
cleavage of the polypeptide chain. Chymotrypsin, a digestive
protease, is produced in inactive form as chymotrypsinogen
in the pancreas and transported in this form to the stomach
where it is activated.
This stops the enzyme from digesting the pancreas or other
tissues before it enters the gut. This type of inactive precursor
to an enzyme is known as a zymogen or proenzyme.
3. Quantity
Enzyme production (transcription and translation
of enzyme genes) can be enhanced or
diminished by a cell in response to changes in
the cell's environment.
This form of gene regulation is called
enzyme induction. For example, bacteria may
become resistant to antibiotics such as penicillin
because enzymes called beta-lactamases are
induced that hydrolyse the crucial
beta-lactam ring within the penicillin molecule.
Another example comes from enzymes in the
liver called cytochrome P450 oxidases, which are
important in drug metabolism.
Induction or inhibition of these enzymes can
4. Subcellular distribution
Enzymes can be compartmentalized, with
different metabolic pathways occurring in
different cellular compartments.
For example, fatty acids are synthesized by one
set of enzymes in the cytosol,
endoplasmic reticulum and golgi and used by a
different set of enzymes as a source of energy in
the mitochondrion, through β-oxidation.
In addition, trafficking of the enzyme to different
compartments may change the degree of
protonation (cytoplasm neutral and lysosome
acidic) or oxidative state [e.g., oxidized (
periplasm) or reduced (cytoplasm)] which in turn
affects enzyme activity.
5. Organ specialization
In multicellular eukaryotes, cells in different
organs and tissues have different patterns of
gene expression and therefore have different
sets of enzymes (known as isozymes) available
for metabolic reactions.
This provides a mechanism for regulating the
overall metabolism of the organism.
For example, hexokinase, the first enzyme in
the glycolysis pathway, has a specialized form
called glucokinase expressed in the liver and
pancreas that has a lower affinity for glucose
yet is more sensitive to glucose concentration.
This enzyme is involved in sensing blood sugar
and regulating insulin production.
Types of inhibition
Competitive
Non-competitive
Uncompetitive
Mixed
Irreversible
Competitive
• A competitive inhibitor and substrate cannot bind to the
enzyme at the same [Link] and inhibitor compete for
binding to the same enzyme.
• Binding is reversible.
• The effect of inhibitor can be over come by increasing the
substrate concentration.
• For example, the drug methotrexate is a competitive
inhibitor of the enzyme dihydrofolate reductase, which
catalyzes the reduction of dihydrofolate to tetrahydrofolate.
• This type of inhibition can be overcome with high substrate
concentration.
• In some cases, the inhibitor can bind to a site other than the
binding-site of the usual substrate and exert an
allosteric effect to change the shape of the usual binding-
site
 Non-competitive
A non-competitive inhibitor binds to a site other
than where the substrate binds.
The substrate still binds with its usual affinity.
However the inhibitor reduces the catalytic
efficiency of the enzyme so that Vmax is
reduced.
In contrast to competitive inhibition, non-
competitive inhibition cannot be overcome with
high substrate concentration.
 Uncompetitive
Uncompetitive inhibition, also known as
anti-competitive inhibition, takes place when
an enzyme inhibitor binds only to the
complex formed between the enzyme and the
substrate (the E-S complex).
In the presence of the inhibitor, the enzyme-substrate
complex is inactive.
This type of inhibition is rare.
Mixed
A mixed inhibitor binds to an allosteric site and the binding of
the substrate and the inhibitor affect each other.
The enzyme's function is reduced but not eliminated when
bound to the inhibitor.
This type of inhibitor does not follow the Michaelis-Menten
equation.
Irreversible
An irreversible inhibitor permanently inactivates the enzyme,
usually by forming a covalent bond to the protein.
Penicillin and aspirin are common drugs that act in this
manner.
 Functions of inhibitors
o In many organisms, inhibitors may act as part of a feedback
mechanism.
If an enzyme produces too much of one substance in the
organism, that substance may act as an inhibitor for the enzyme
at the beginning of the pathway that produces it, causing
production of the substance to slow down or stop when there is
sufficient amount.
This is a form of negative feedback.
Major metabolic pathways such as the citric acid cycle make
use of this mechanism.
o Since inhibitors modulate the function of enzymes they are
often used as drugs.
Many such drugs are reversible competitive inhibitors that
resemble the enzyme's native substrate, similar to methotrexate.
other well-known examples include statins used to treat high
cholesterol, and protease inhibitors used to treat retroviral
infections such as HIV.
A common example of an irreversible inhibitor that is used
as a drug is aspirin, which inhibits the COX-1 and COX-2
enzymes that produce the inflammation messenger
prostaglandin.
o Other enzyme inhibitors are poisons.
For example, the poison cyanide is an irreversible enzyme
inhibitor that combines with the copper and iron in the active
site of the enzyme cytochrome c oxidase and blocks
cellular respiration.
Factors effecting enzyme activity
1. Substrate concentration
2. Enzyme concentration
3. Temperature
4. pH
1. Substrate concentration
- The velocity of enzyme catalysed reaction is
directly related with the substrate concentration.
- The enzyme catalysed reaction involves
reversible formation of enzyme substrate
complex due to binding of substrate(S) to
enzyme (E) to form (ES) which spontaneously
break down to product (P) and enzyme(E)
becomes free again.
 E + S  ES E + P
At the beginning of the reaction, since no product
(P) is present ES formation from P is zero.
As the [S] is in large excess of [E], all the [E] is
assumed to be in as ES(the enzyme substrate
concentration) and the product formation is
dependent on the rate of
disappearance(conversion) of ES.
Substrate concentration: Enzymic
reactions

Vmax

Reaction
velocity

Substrate concentration
 Faster reaction but it reaches a saturation point
when all the enzyme molecules are occupied.
 If you alter the concentration of the enzyme then
Vmax will change too.
© 2007 Paul Billiet ODWS
2. Enzyme concentration
Effect of Substrate on Enzyme Activity
Effect of pH on enzyme activity
Enzyme works best within a narrow
pH range
Each enzyme works best at particular
pH, known as its optimum pH level.
At extreme pH levels, enzymes lose
their shape and function and become
denatured.
The effect of pH
Extreme pH levels will produce
denaturation
The structure of the enzyme is changed
The active site is distorted and the
substrate molecules will no longer fit in
it
At pH values slightly different from the
enzyme’s optimum value, small changes
in the charges of the enzyme and it’s
substrate molecules will occur
This change in ionisation will affect the
binding of the substrate with the active
Effect of pH on enzyme activity
 The effect of pH
Optimum pH values

Enzyme
activity Trypsin

Pepsin

1 3 5 7 9 11

© 2007 Paul Billiet ODWS

pH
The effect of temperature
Q10 (the temperature coefficient) =
the increase in reaction rate with a 10°C
rise in temperature.
For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or
triples with every 10°C rise in
temperature)
Enzyme-controlled reactions follow this
rule as they are chemical reactions
BUT at high temperatures proteins
denature
The optimum temperature for an
enzyme controlled reaction will be a
balance between the Q10 and
denaturation.
0°C
Low temperatures  low Kinetic
Energy of enzymes and substrates.

No/Very few enzyme-substrate

complexes are formed.

Enzymes are inactivated.

 20°C (increasing temperature)
Increasing the temperature will lead to the increase
in kinetic energy of enzyme and substrate molecules.

Enzyme and substrate molecules move with

increasing speed and collide more frequently with
each other.

This increases the rate of enzyme-substrate complex

formation This increases the rate of enzyme-
substrate complex formation and product formation.

Rate of reaction
increases
37°C
As the temperature continues to
increase, the rate of enzyme activity
also increases until the optimal
temperature is reached.

Optimal temperature is the

temperature at which the enzyme
works best. Rate of product formation
is highest!
Beyond Optimal Temperatures
At high temperatures (>60°C), weak bonds within the
enzyme molecule are broken
Enzyme loses its shape and its active site.
Loss of shape leads to a loss of function. Enzyme is said
to have denatured
Denaturation is the change in 3D structure of an enzyme
or any other protein caused by heat or chemicals such as
acids or alkali, causing it to lose its function.
 Denaturation

Different enzymes denature at different temperatures. Most enzymes denature a

mperatures higher than 60°C. However, there are some enzymes that stay active
at high temperatures like 80°C (Enzymes in the bacteria Thermus aquaticus)
 The effect of temperature

Q10 Denaturation
Enzyme
activity

0 10 20 30 40 50
Temperature / °C

© 2007 Paul Billiet ODWS

 The effect of temperature
For most enzymes the optimum
temperature is about 30°C
Many are a lot lower,
cold water fish will die at 30°C because
their enzymes denature
A few bacteria have enzymes that can
withstand very high temperatures up to
100°C
Most enzymes however are fully
denatured at 70°C
© 2007 Paul Billiet ODWS
Specificity of enzymes
 Enzymes are highly specific. An enzyme may have
1. Reaction specificity - a substrate can undergo many reaction but a
particular enzyme catalyses only one of the various reaction.
2. Substrate specificity - the degree of specificity may vary from
enzyme to enzyme.
a. Absolute specificity - is rare in which enzyme dose not bind with
any other substrate. ex - urease is absolutely specific for urea
b. Relative specificity - for substrate may be group dependent or
bond dependent
i. Group dependent - is found in enzymes like trypsin and
chymotripsin. Tripsin hydrolysis the residues of only lysine and
arginine. While chymotrypsin hydrolysis residues of only
aromatic amino acids.
ii. Bond specific- is found in case of proteolytic enzymes,
glycosidases and lipases, which act on peptide bonds, glycosidic
bonds and ester bonds respectively.
Mode of Action

Substrate fits in the enzyme active site,

just like a key fits into a lock.

An enzyme-substrate complex is formed.

Chemical reactions occur at the active site and products

are formed.
Introduction -
 The basic mechanism by which enzymes catalyze
chemical reactions begins with the binding of the
substrate (or substrates) to the active site on the
enzyme.
 The active site is the specific region of the enzyme
which combines with the substrate.
 The binding of the substrate to the enzyme causes
changes in the distribution of electrons in the chemical
bonds of the substrate and ultimately causes the
reactions that lead to the formation of products.
 The products are released from the enzyme surface to
regenerate the enzyme for another reaction cycle.
 The active site has a unique geometric shape that is
complementary to the geometric shape of a substrate
molecule, similar to the fit of puzzle pieces.
 This means that enzymes specifically react with only one
or a very few similar compounds.
Lock and Key Theory:
The specific action of an enzyme with a single
substrate can be explained using a Lock and Key
analogy first postulated in 1894 by Emil Fischer.
In this analogy, the lock is the enzyme and the key
is the substrate.
Only the correctly sized key (substrate) fits into
the key hole (active site) of the lock (enzyme).
Smaller keys, larger keys, or incorrectly positioned
teeth on keys (incorrectly shaped or sized substrate
molecules) do not fit into the lock (enzyme).
Only the correctly shaped key opens a particular
lock.
Not all experimental evidence can be adequately
explained by using the so-called rigid enzyme
model assumed by the lock and key theory. For
this reason, a modification called the induced-fit
theory has been proposed.
The induced-fit theory assumes that the
substrate plays a role in determining the final
shape of the enzyme and that the enzyme is
partially flexible.
This explains why certain compounds can bind to
the enzyme but do not react because the
enzyme has been distorted too much.
Other molecules may be too small to induce the
proper alignment and therefore cannot react.
Only the proper substrate is capable of inducing
the proper alignment of the active site.
 The induced fit theory assumes that the substrate plays a role in
determining the final shape of the enzyme is partially flexible.
 This explains why certain compounds can bind to the enzyme but
not react because the enzyme has been distorted too much.
 Other molecules may be too small to induce the proper alignment
and therefor cannot react. Only the proper substrate is capable of
inducing the proper alignment of the active site.