UNIT – I

Ferritin:

(i) Role of ferritin:

Iron is stored mostly in ferritin, an iron storage protein. For
the production of hemoglobin, myoglobin, and cytochrome, ferritin
release iron is required.
This is important because unbound free iron is extremely
toxic and causes cellular damage by promoting the production of free
radicals.
Found in liver, spleen, and bone marrow, ferritin is
significant iron storage.

(ii) Structure of Ferritin:

Inside and outside of cells, ferritin is an iron-binding protein.
Apoferritin, creates a roughly spherical container within which ferric iron is kept
as the ferrihydrite mineral ferric iron (Apoferritin refers to the iron-free form of
the protein; their on-containing form is termed holoferritin or simply ferritin).
There are 24subunits in the apoferritin shell. The two sorts of subunits
are H and L. The ratio of these subunits varies a lot depending on the tissue type,
and it can alter a lot under inflammatory and infectious situations.
H- subunit-rich tissue ferritins (found primarily in the heart and kidney)
to L-subunit-rich tissue ferritins (found predominantly in liver and spleen).
Each apoprotein molecular mass of around 450,000d.The L monomer is
made up of 174 amino acids and has a molecular weight of 174.
The H monomer has a molecular mass of 21,000 d and is made up of 182
amino acids.
A protein having 24 subunits of 500k Da. It binds 4500 iron molecules
which accounts for a larger iron storage.

Its measurement is used to assess iron stores in the body. Ferritin can be
measured by RIA (RADIO IMMUNO ASSAY) , ELISA ( ENZYME LINKED
IMMUNOSORBENT ASSAY) and Chemiluminescence.
Transferrin:
Several structurally important and related Fe- proteins, all of
which are glycoprotein’s, makeup transferrins. Their molecular weights
(molar masses) are approximately 80 k Da.
New hemoglobin is produced in the bone marrow, while old red
cells are destroyed in the spleen and liver.
Transferrins [as a class of protein, serum protein, lactoferrin
(milk), and ovatransferrin (egg)] are iron binding proteins that transport
iron in higher animals via the bloodstream to the site of synthesis of other
iron-containing compounds such as hemoglobin, cytochrome, and
others, where it is inserted into the porphyrin ring via enzyme.
Iron binds to transferrin for transportation in plasma after re-absorption.
Whereas the storage occurs in hepatoparenchymal cells, reticuloendothelial cells,
bone marrow, liver and spleen.

Each transferrin molecule binds to two iron atoms .Transferrin transports iron
to various organs and tissues.

Serum iron + amount of iron bound to transferrin = total iron in circulation.

Determination of transferrin can provide total iron binding capacity.

Transferrin can be measured by RIA, ELISA and Chemiluminescence.

Sidorphores:
Sidorphores are compounds from ancient Greek words sidor
‘iron’ and phore ‘carriers’ meaning iron carriers.
Sidorphores are low molecular weight iron chelating agents
compounds produced by ‘rhizospheric bacteria’ under iron limited
conditions.
They are small, with a high affinity for ferric iron chelating
compounds.
Sidorphores are produced by microorganisms under
restricted iron conditions.
Many sidorphores produced by bacteria and fungi are strong
enough to remove iron from host-binding proteins.
Sidorphores usually form a stable hexahendate, octahedral
complex with Fe3+.
Iron is a constituent of protein. It activates the number of enzymes.
It plays an essential role in the nucleic acid metabolism. It is
necessary for synthesis and maintenance of chlorophyll in plants.
Sidorphores are amongst the strongest soluble Fe3+ binding agents.
Sidorphores are produced by both pathogenic and non
pathogenic bacteria in different environments.
Siderophores produced by microorganisms are structurally
very diverse with molecular weights of 150 up to 2000Da.

The siderophore is a chelating ligand that provides Fe(III) with

a set of oxygen donors, and the complexes are considered to have
 six coordinates (III).
Calcium signaling proteins:
Calcium signaling is the use of calcium ions (Ca2+) to
communicate and drive intracellular processes often as a step in signal
transduction.
Ca2+ is important for cellular signalling, for once it enters
the cytosol of the cytoplasm it exerts allosteric regulatory effects on
many enzymes and proteins.
Ca2+ can act in signal transduction resulting from activation
of ion channels or as a second messenger caused by indirect signal
transduction pathways such as G protein-coupled receptors.
Concentration Regulation:
The resting concentration of Ca2+ in the cytoplasm is
normally maintained around 100 nm. This is 20,000- to 100,000-fold
lower than typical extracellular concentration.
To maintain this low concentration, Ca2+ is actively pumped
from the cytosol to the extracellular space, the endoplasmic
reticulum (ER), and sometimes into the mitochondria.
Certain proteins of the cytoplasm and organelles act as
buffers by binding Ca2+. Signaling occurs when the cell is stimulated to
release Ca2+ ions from intracellular stores, and/or when Ca2+ enters the
cell through plasma membrane ion channels.
Under certain conditions, the intracellular Ca2+ concentration
may begin to oscillate at a specific frequency.
Roles of calcium signalling:
Calcium signaling is essential for the regulation of a diverse
set of crucial functions within the human body, such as overseeing cell
death, gene transcription, muscle contraction, exocytosis, neuronal
transmission, cell motility (including the movement of flagella and
cilia), fertilization, cell growth and proliferation, neurogenesis, synaptic
plasticity, secretion of saliva, enzyme activity, and more.
Calcium is a ubiquitous intracellular messenger that can
govern diverse cellular functions because it, directly and indirectly,
modulates a myriad of different proteins that are involved in different
jobs.
Transcription factors such as NF-AT, kinases, and
phosphatases, and calcium-binding protein calmodulin (CaM) are all
impacted by calcium activity.

Metalloenzymes:
Metalloenzymes are involved in several different reactions
and the metal can display distinct roles, such as redox chemistry or
substrate activation.
The activation mechanism is mainly described to occur by
bond polarization upon coordination to the metal center.
A metalloenzyme is an enzymatic protein in which a metal as
metal ion is embedded in the cavity of the enzyme and forms strong
bonds with the donor atoms of the protein.
The donor atoms of proteins may be either soft base as sulphur
or hard bases such oxygen and nitrogen. In the similar way the metals
may be either soft metal such as Cu+, Hg+ and Cd+ or hard such as
Fe3+ and Zn2+.
The protein part is called as an apoenzyme and a metal ion
or complex metal ion is called a prosthetic group.
• metal is firmly bound
• metal to protein ratio is constant
• metal to enzyme activity ratio is constant
• metal is unique
• no enzyme activity without metal
Examples of metalloenzymes:
– superoxide dismutase (Zn and Cu)
– carboxypeptidase A (Zn)
– carbonic anhydrase (Zn)
– cytochrome oxidase (Fe and Cu)
– xanthine oxidase (Co and Fe)
Metal-activated enzymes :
• metal is reversibly bound
• metal to protein ratio is variable
• metal to enzyme activity ratio is variable
• metal is not necessarily unique
 • enzyme activity may exit without metal.

Metal-activated ezymes
• Examples of metal-activated enzymes – creatine kinase
(Mg, Mn, Ca or Co) – glycogen phosphorylase kinase (Ca) – salivary
and pancreatic alpha-amylases (Ca)
Enzymes:
Enzymes are large protein molecules that catalyze large
number of biochemical reactions. They increase the rate of biochemical
reactions about 106 times compared to the uncatalyzed rate.
They lower the activation energy for the formation of one
product rather than other and therefore are highly specific.
Zinc enzymes:
Zinc has a highly concentrated charge in comparison to its
relatively small ionic radius (0.65A°) and binds modestly to anions such
as carboxylates and phosphates.
Its second characteristic is its high affinity for electrons,
making it a strong Lewis acid, similar to copper and nickel.
It does not show variable valence, which might lead to it
being preferred quite simply because it does not introduce the risk of
free radical reactions.
Zinc is the second most abundant trace element in the human
body. An average adult has about 3 g of Zn, corresponding to a
concentration of zinc of about 0.6 mM, most of which (some 95%) is
intracellular.
Zinc is essential for growth and development in all forms of
life, has been proposed to have beneficial therapeutic and preventative
effects on infectious diseases, including a shortening of the length of the
common cold in man.
Zinc is found in more than 300 enzymes, where it plays both
a catalytic and a structural role. It is the only metal to have
representatives in each of the six fundamental classes of enzymes
recognised by the International Union of Biochemistry.
Carboxypeptidase:
Reaction:
The metal ion is coordinated to two N-atoms histidine
residues, to an oxygen atom of a glutamate residue that acts as
bidentate ligand and to a water molecule.
The cavity has a hydrophobic pocket close to Zn2+ ion that
can accommodate organic group of the peptide undergoing
hydrolysis.
The site of cleavage is specific in two ways: it occurs at the
C-terminal amino acid and it exhibits a high selectivity for substrates
in which the C-terminal amino acid contains a large aliphatic or
aromatic side chain.

The carbonyl group of the substrate hydrogen bonds to an

arginine (Arg-145) and the Zn2+ ion bonds to the oxygen of the
peptide carbonyl group.
The Arg-127 bonds to oxygen of carbonyl group of
peptide of substrate and the phenolic group of Try-248 residue
hydrogen bonds to-NH group of peptide substrate.
Structure of Carboxypeptidase A:

The enzyme consists of a single protein chain of 307 amino

acids and one Zn2+.
Molar mass: 34800
Zn2+ ion tetrahedrally coordinated to:Two Histidine N
atoms,An oxygen atom from carboxyl side chain of glutamic
residue,One water molecule.

1-The tyrosine side chain of the substrate binds to the non-polar pocket
in the active site of the enzyme.
2-The NH- hydrogen of the peptide bond to be cleaved is hydrogen
bonded to the OH group of tyrosine
 248.
3-The negatively charged terminal carboxylic group of glycyltyrosine
(substrate) interacts electrostatically with the positively charged side
chain of arginine 145
4-The carboxyl oxygen of the peptide bond to be cleaved is coordinated
to the zinc ion.
5-The terminal amino group of the peptide chain is hydrogen bonded
through the water molecule to the side chain of glutamate 270.

Carbonic Anhydrase:
Carbonic anhydrous is defined as an enzyme. It is found
in red blood cells, gastric mucosa, pancreatic cells, and renal tubules
that catalyzes the inter conversion of carbon dioxide (CO2) and
carbonic acid (H2CO3).
Carbonic anhydrase plays an important role in respiration by
influencing CO2 transport in the blood. This enzyme also functions in
the hydrochloric acid formation by the stomach.
Carbonic anhydrase enzyme helps to maintain acid-base
homeostasis, fluid balance and regulate pH. Based on its location, the
enzyme role changes slightly. For example, carbonic anhydrase forms
acid in the stomach lining.
Structure and Function:
Many forms of carbonic anhydrase take place in nature.
The zinc ion can be coordinated by the imidazole rings of three histidine
residues, His94, His96, and His119, in the best-studied, -carbonic
anhydrase shape, which is present in animals.
The major enzyme function in animals is to
interconvert the bicarbonate and carbon dioxide to maintain acid-base
balance in the blood and other tissues and also to help transport carbon
dioxide out of the tissues.
Reaction:
The following reaction illustrates the catalysis of carbonic
anhydrase in our tissues:
CO2 + H2O → H2CO3 → H+ + HCO-3
Catalyzation of the carbonic anhydrase in the lungs is
represented by:
H+ + HCO-3 → H2CO3 → CO2 + H2O
The reaction's reason being in the opposite directions for
lungs and tissues is because of the variable pH levels found in them.
Without the carbonic anhydrase catalyst, however, this reaction is much
slower; with the catalyst, the reaction is 107 times faster.
The reaction, which is catalyzed by the carbonic anhydrase is
given by:
HCO-3 + H+ ⇋ CO2 + H2O
Since carbonic acid has a pKa of up to 6.36 (depending on
the medium), a lower percentage of the bicarbonate can be protonated at
pH 7.
Carbonic anhydrase is the fastest enzyme, and its rate is
normally limited by the rate at which its substrates diffuse. The typical
catalytic rates of the various forms of this enzyme range from 104 -106
reactions per second.
An anhydrase can be defined as an enzyme, which catalyzes
the water molecule removal from a compound, and so, it is the "reverse"
reaction, which gives the carbonic anhydrase its name because it
removes water a molecule from the carbonic acid.
Carbonic anhydrase in the lungs transforms bicarbonate into
carbon dioxide, which is ideal for exhalation.
Mechanism:
The Zn2+ ion is more acidic in carbonic anhydrase than in
carboxypeptidase. The presence of a neutral or less basic histidine
residue instead of the glutamate residue contributes to the greater acidity
of Zn2+ ion.
The three histidine residues are pulled back; therefore
Zn2+ ion becomes more electronegative and more acidic towards the
fourth position. Thus, the coordinated water becomes more polarized
and losses H+ ion to give Zn-OH–.
The nucleophilic OH– then attacks on the carbon atom of
CO2 captured in the hydrophobic pocket near the Zn2+ ion, and a
transient five coordinated Zn2+ ion is formed in which a carbonato
oxygen from HCO3– coordinates to the Zn2+ ion.
After rearrangement, the HCO3– ligand is replaced by H2O.
The protonation of H2O coordinated to Zn2+ ion then regenerate Zn-
OH– which then attacks another CO2 with the continuation of the
catalytic cycle.
Iron Enzymes:
Catalase and peroxidase enzymes:
Catalase are heme enzymes that catalyze reactions of hydrogen
peroxide. In catalase, the enzymatic reaction is the disproportionation of hydrogen
peroxide and the function of the enzyme appears to be prevention of any buildup of
that potentially dangerous oxidant .
2H2O2 −−−−−> 2H2O+O2
Peroxidase reacts by mechanisms similar to catalase, but the reaction
catalyzed is the oxidation of a wide variety of organic and inorganic substrates by
hydrogen peroxide.
 H2O2 +AH2 −−−−−>2H2O +A
The catalase reaction can be seen to be a special case of in which the
substrate, AH2, is hydrogen peroxide.
Some examples of peroxidases that have been characterized are
horseradish peroxidase, cytochrome c peroxidase, glutathione peroxidase, and
myeloperoxidase.
Mechanism:
The accepted mechanisms for catalase and peroxidase are described in
Reactions
FeIII(P)++ H2O2→FeIII(P)(H2O2)+→FeIV(P−)(O)++ H2O
Compound I
catalase:
FeIV(P⋅−)(O)++ H2O2→FeIII(P)++ H2O +O2
Compound I
peroxidase:
FeIV(P⋅−)(O)+ +AH2→FeIV(P)(O)+HA⋅+H+
Compound I Compound II
FeIV(P)(O)+AH2→FeIII(P)+ +HA+OH--
Compound II
2HA→A+AH2
In the catalase reaction, it has been established by use of H2O2 that the
dioxygen formed is derived from hydrogen peroxide, i.e., that O—O bond cleavage
does not occur in , which is therefore a two-electron reduction of compound I by
hydrogen peroxide, with the oxo ligand of the former being released as water.
For the peroxidase reaction under physiological conditions, it is
believed that the oxidation proceeds in one-electron steps , with the final formation
of product occurring by disproportionation or coupling of the one-electron
oxidized intermediate.

COPPERENZYME:
Superoxide dismutase(SOD) a copper enzyme:
Superoxide dismutase is a catalytic enzyme that catalyses the
elimination of the harmful superoxide anion, O2-, which is produced as a
byproduct of oxidative metabolism. Superoxide is converted to molecular
 oxygen and hydrogen peroxide by this enzyme. The subsequent work of
enzymes like catalase removes hydrogen peroxide, which is a potentially
hazardous chemical. As a result of their collaboration, SOD and catalase
help to safeguard organisms that use di oxygen from potentially
hazardous byproducts of O2metabolism.

 Superoxide dismutase (SOD) is present in all aerotolerant organisms

for the purpose of minimizing the concentration of superoxide, O2,
and thus providing protection against oxygen toxicity.

 The SOD like, Ni SOD, either Fe or Mn SOD, which seem to be the

same protein, and Cu/Zn SOD.

 Fe or Mn SOD is found in the prokaryotes or the mitochondria of

eukaryotic cells, while Cu/Zn SOD is found in the cytoplasm of
eukaryotic cells.

There are three type of superoxide dismutase :

(1) Copper-Zinc superoxide dismutase, Cu Zn SOD

(2) Manganese superoxide dismutase, Mn SOD

(3) Iron superoxide dismutase, Fe SOD

Structure of Cu-Zn superoxide dismutase:

Cu SOD (bovine superoxide dismutase) is present in the

mitochondria of eukaryotic cells, while the other two are found in
bacteria (prokaryotes). Cu2+ and Zn2+ ions are coupled to the imidazole of
a histidine residue, according to a Cu Zn SOD crystal structure
determination(fig. 2). The Cu2+ ion is a deformed square pyrimidal site
bound to four imidazole histidine nitrogen atoms and a water molecule,
whereas the Zn2+ ion is tetrahedrally coordinated to three imidazole
 histidine nitrogen atoms and oxygen from the asparatate residue.

Fig.2.14structureofCu-Znsuperoxidedismutase
The molar mass of copper-zinc dismutase is around
16,000. The Cu2+ ion has been demonstrated to be the function alone,
where as the Zn2+ ion serves as a structural stabilizer by holding the
bridging imidazole histidine residue in place.

Cu 2+ is a more important ion that cannot be substituted

by another metal while maintaining activity. The Zn 2+ ion, on the other
hand, can be replaced with other divalent metals such as Co or Cd while
maintaining the majority of the activity.

Mechanism Cu-Zn superoxide dismutase

The following point maybe noted:

When Cu(II) and Zn(II) are removed from an enzyme, its activity is
diminished. Only the addition of Cu(II) reactivates the enzyme. As a result, the
role of Zn(II) is only relevant from a structural stand point. Further more,
Zn(II)occupies all four coordination sites in Cu-ZnSOD. The displacement of
His 46 by superoxide is thought to be the mechanism by which it binds to
 Cu(II).

 It has been proposed that the imidazole bridge breaks and reforms
during each catalytic cycle of the reaction.
 The pKa of the other nitrogen atom is lowered when a histidine ring
nitrogen atom is coordinated to Zn(II). As a result, it prefers to attach
a proton rather than Cu (I).
 When a proton is transferred to a peroxide ion, which is then
transformed to H2O2, the bridge is reformed. This idea also creates a

location at the Cu(I)centre for substrate(O2-) binding, eliminating the

necessity for a potentially high-energy five-coordinate transition
state.