3.

3 BIOCHEMISTRY OF ZINC AND COPPER:

STRUCTURE AND FUNCTIONS

Carbonic anhydrase

Carbonic anhydrase (contains Zn) - which catalyses the rapid inter conversion of
CO2 and water to bicarbonate and proton.
Carbonic
CO2 H2O anhydrase HCO3- H+

In the absence of carbonic anhydrase and at pH 7 the above reaction is very slow,
the rate constant being 10-3 s-1. The turnover of carbon dioxide by biological
systems is very high and a reaction with such a small rate constant cannot serve
the purpose. In presence of carbonic anhydrase, however the hydration of carbon
dioxide proceeds with a rate constant of 106 s-1, making it one of the fastest
enzymatic reactions.

The reverse reaction,

HCO3- H+ H2CO3 CO2 H2O

This involves dehydration of carbonic acid, is also catalyzed by carbonic

anhydrase. For this reason the latter is often referred to as carbonic dehydratase.

The crystal structure of carbonic anhydrase H H

revels that the zinc (II) ion is placed in a O
conical cavity about 16 pm deep, which is
lined up with several histidine residues. The Zn
zinc (II) ion is coordinated to three histidine
N
ligands through nitrogen atoms and to one N
N
water molecule through oxygen atom. One HN
NH
such unit has a molecular weight of
N
approximately 30000. The skeletal structure H
which has a near tetrahedral geometry is Histidine residues
shown below.
 Dr. Saritha Chandran A.
Department of Chemistry and Center for Research

The mechanism of the reaction catalyzed by carbonic anhydrase is outlined

below.
N
N Zn OH
H2 O
-H+ N
(b)

N
N H N Zn O H
N Zn O N H O
N (a) H (c) H

-HCO3-
O O C O
N

O C
N Zn O H
N N H O
OH
N Zn (d) H
N
O H
O
(f) H
C O
N
N Zn O H
N H O
(e) H
a) The attachment of three histidine nitrogen’s to zinc (II) ion induces a
positive charge on the oxygen atom of the aqua ligand.
b) This results in a loss of proton, so that instead of a water molecule, a
hydroxide group now occupies the fourth coordination site of zinc.
c) For this reason the mechanism is called zinc-hydroxide pathway. Another
water molecule becomes attached to the hydroxyl group through hydrogen
bonding.
d) A CO2 molecule coordinates with intermediate (c). In this union, the carbon
of carbon dioxide becomes attached to the oxygen of hydroxyl group, while
one of the oxygen atoms of CO2 becomes attached to the proton of the
hydrogen- bonded water molecule.
e) Next the bond between O of the OH group and C of CO2 strengthens at the
cost of the bond between O and H of the OH group (c). The intermediate
(c) rearrange so that in transition state.
f) Zinc is 5 coordinated, being firmly bound to three histidine groups and
loosely to a bicarbonate group and an aqua ligand. Finally the bicarbonate
anion dissociates and the cycle starts once again. The net reaction is a
release of bicarbonate anion and a proton. The mechanism may proceed in
clockwise or anti-clockwise direction depending on the requirements of the
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biological systems.

St. Teresa’s College (Autonomous), Ernakulam, Kerala

[email protected], https://sites.google.com/view/sarithachandrana/academics
 Dr. Saritha Chandran A.
Department of Chemistry and Center for Research

The reaction catalyzed by carbonic anhydrase produce protons and bicarbonate

ions (alkali). The reverse reaction, in which protons and bicarbonate ions are
consumed, is also catalyzed by carbonic anhydrase. Thus the primary function of
the enzyme is to maintain acid-base balance in blood and other tissues. Yet
another function is to transport CO2 out of the tissues.

Carboxypeptidase A

Carboxypeptidase, an enzyme synthesized in the pancreas and thereafter secreted

into small intestine catalyses the hydrolysis of proteins that are assumed as
components of food into amino acids. During the hydrolysis reaction, a water
molecule acts on the amide linkage of the protein unit and cleavs the carbonyl
terminal of amino acid of the peptide chain.

H2O
Pro Leu Glu Phe Pro Leu Glu Phenyl alanine
Carboxypeptidase A

R O R' O R''
H2O
CH C N C NH C COOH
H
Carboxy peptidase

R O R' R''

CH C NH C COOH H2N C COOH

H H

The enzyme consists of a protein chain of 307 amino acid residues plus one Zn2+
ion to yield a MW of about 34,600. The molecule is roughly egg shaped with
maximum dimension of approximately 5000 pm and a minimum of 3800 pm.
There is a cleft on one side that contains the zinc ion, the active site. The metal is
co-ordinates approximately tetrahedrally to –nitrogen atoms and an oxygen atom
from 3- amino acids (His 69, Glu 72 and His 169) in the protein chain.
2
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St. Teresa’s College (Autonomous), Ernakulam, Kerala

[email protected], https://sites.google.com/view/sarithachandrana/academics
 Dr. Saritha Chandran A.
Department of Chemistry and Center for Research

H H
O

Zn

Histidine N N
O
NH
N O C
H Histidine
H2C

Glutanic acid

The 4th coordination site is free to accept a pair of electron from a donor atom in
the substrate to be cleaved. The enzyme is thought to act through co-ordination
of the live atom to the carbonyl group of the amide linkage. In addition a nearby
hydrophobic pocket envelops the organic group of the amino acid to be cleaved
and those amino acids with aromatic side groups react most readily.
Accompanying these events is a change in conformation of the enzyme. The
argentine side chain moves about 200 pm closer to the carboxylate group of the
substrate and the phenolic group of the tyrosine comes within the hydrogen
bonding distance of the imido group of the C-terminal amino acid, a shift of 1200
pm. The hydrogen bonding to the free carboxyl group (by argentine) and the
amide linkage (by tyrosine) not only holds the substrate to the enzyme but helps
breaks the N—C bond. Nucleophilic displacement of the amide group of
attacking carboxylate group from glutamate group could form an anhydride link
to the remainder of the peptide chain. Hydrolysis of the anhydride could then
complete the cycle and regenerate the original enzyme. More likely, the glutamate
acts indirectly by polarizing a H2O molecule that attacks the amide linkage (N—
C linkage).

The protein performs a host of functions which are essential for the life processes.
The biological system of humans, as well as of other organism has the capacity
to biosynthesize the requisite proteins from amino acids the source of amino acids
are proteins that are consumed as part of the diet.

The main function of carboxypeptidise is to hydrolyze such proteins, so as to

clean their amino acids. The latter can then be used to synthesize the proteins
which the body actually requires. Carboxypepetidase also assists in blood clotting
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and wound healing. Moreover it participates in the biosynthesis of insulin.

St. Teresa’s College (Autonomous), Ernakulam, Kerala

[email protected], https://sites.google.com/view/sarithachandrana/academics
 Dr. Saritha Chandran A.
Department of Chemistry and Center for Research

Structure of CPA active site

Active site

H
Zn
N
N N
His 69 O N H

O C His 169
CH2 CH2 H2C
Glu 72

Protein portion of the Enzyme

The 3-amino acid residues (Arg.145, phenolic OH of Tyr – 248 and carboxylic
end of Glu – 270) are playing some important role in the enzymatic activity of
CPA.

CPA is released from their inactive precursors or Zymogens (ie, procarboxy

peptidases) in the pancreas for the digestion of proteins. CPA exists in different
forms (ie, CPA–α, CPA–β, CPA–γ etc) depending on the size of the fragment
lost from the zymogen. CPA–α is commonly known as CPA. If contains 307
amino acids. CPA–β contains 305 amino acids and CPA–γ contains 300 amino
acids.

Superoxide dismutase (SOD)

Several powerful oxidants are produced during metabolic process both in blood
cells and other cells of the body. These include superoxide, hydrogen peroxide,
peroxy radicals and hydroxyl radicals. Reactions and chemicals which generate
these toxic oxidants are called pro-oxidants. On the other hand, reactions and
compounds which dispose of these species are called anti-oxidants and these
include NADPH, GSH, ascorbic acid and vitamin E. in a normal cell, there is an
appropriate pro oxidant: antioxidant balance. Superoxide dismutase, catalyze and
glutathione protect blood cells from oxidative stress and damage.
4
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St. Teresa’s College (Autonomous), Ernakulam, Kerala

[email protected], https://sites.google.com/view/sarithachandrana/academics
 Dr. Saritha Chandran A.
Department of Chemistry and Center for Research

Superoxide dismutase catalyses the rapid removal of the toxic superoxide anion
(O2-), the by-product of oxidative metabolism. The enzyme catalyzes the
conversion of superoxide to molecular oxygen and hydrogen peroxide.

Cytochrome C oxidase and may other enzymes can convert O2 to H2O without
the formation of the toxic intermediates. SOD is an enzyme that alternately
catalyses the dismutatin (or partitioning) of the superoxide (O2-) radical into either
ordinary molecular O2 or H2O2. Superoxide is produced as a byproduct of oxygen
metabolism and if not regulated causes many types of cell damage. SOD is an
important antioxidant defense in nearly all living cells exposed to O2.

SOD enzymes deal with the superoxide radical by alternately adding or removing
an electron from the superoxide molecules it encounters, thus changing O 2- into
one of two less damaging species either molecular O 2 or hydrogen peroxide
(H2O2). Hydrogen peroxide, itself a potentially harmful substance is rapidly
removed by the subsequent action of enzymes such as catalase. Thus working
together SOD and catalase help in protecting the organism which utilize dioxygen
from potentially toxic by-products of O2 metabolism.

O2

2O2- 2H+ H2O2 2H2O O2

Superoxide Catalase
dismutase

3- types of SOD are known. The first characterized is Cu, Zn containing SOD
which exists in eukaryotic cells. In micro organisms Fe-SOD and Mn-SOD are
found. Cu-Zn SOD is quite stable than later SOD’s because it does not release
toxic metal Cu2+ or Cu+. Mn-SOD and Fe-SOD have distorted Trigonal-
bipyramidal structure in which metal is four coordinated to three histidine
residues and one tyrosine residue.

H2O
N-His N-His
Asp-O Fe Asp-O Mn
N-His N-His
5

N-His N-His
Page

St. Teresa’s College (Autonomous), Ernakulam, Kerala

[email protected], https://sites.google.com/view/sarithachandrana/academics
 Dr. Saritha Chandran A.
Department of Chemistry and Center for Research

The SOD catalyzed dismutation of superoxide may write for CU-Zn SOD as
follows;

1. Cu2+—SOD + O− +
2 ⟶ Cu — SOD + O2

2. Cu2+—SOD + O− + 2+
2 + 2H ⟶ Cu —SOD + H2 O2

The general form applicable to all the different metal-coordinated forms of SOD
can be written as follows;

M(n+1)+ SOD O2- Mn+ SOD O2

Mn+ SOD O2- 2H+ M(n+1)+ SOD H2O2
Where M = Cu (n=1); Mn(n = 2); Fe (n = 2) + Ni (n = 2)

Mn-SOD is regarded as a potential treatment in a number of medical conditions,

e.g. Oxygen-damaged retinal. The SODs are also believed to have an anticancer
role.

Structure of Cu-Zn SOD

Cu-Zn SOD is normally purified from bovine RRC. It is a homodimer of mw

32,500. It is dimerise and represented as Cu2—Zn2—BSOD. It contains two
subunits at the active site of each monomeric unit, one containing one Cu site
while the other containing one Zn-site. These two subunits are bridged through a
deprotonated imidazole ring of histidyl chain (His-61), in the resting condition;
Cu (II) is almost in a square planar geometry, which Zn (II) is in tetrahedral
geometry. In fact Cu (II) is equatorially coordinated and 4-histidyl imidazole (His
-46, His-118, His-44 and His 61) and a H2O molecule remains weakly bound to
one axial position to give a highly distorted square pyramidal geometry. The Zn
(II) centre is coordinated to 3-histidyl imidazoles (His-78, His-69 and bridging
His -61) and the carboxylate group of an aspartyl residue (Asp-81) to attain the
tetrahedral geometry.
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St. Teresa’s College (Autonomous), Ernakulam, Kerala

[email protected], https://sites.google.com/view/sarithachandrana/academics
 Dr. Saritha Chandran A.
Department of Chemistry and Center for Research

61 His

OH2 O2- channel

(Asp - 81) O (II)
(His - 78) N Zn N N N (His-118)
(His - 89) N
Cu
His - 44 N (II)
N His- 46)

In Cu-Zn SOD, the role of Zn (II) is structural only. Zn can be replaced by other
divalent metals with the retention of most of the catalytic activity. But the
presence of Cu (II) is essential for SOD activity. It has been found that the Zn-
depleted protein lacks in thermal stability and it denatures at lower temperature
than the native protein.

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St. Teresa’s College (Autonomous), Ernakulam, Kerala

[email protected], https://sites.google.com/view/sarithachandrana/academics