Smaranika Bardhan

PG-2, Sem-4
Inorganic chemistry
2017
Contents
 Introduction

 Structural features

 Biological function of CA

 Characteristics of enzymatic activity

 application
 Carbonic anhydrase is a Zn(II) containing metalloenzyme

 This enzyme was first identified in 1933, in red blood cells of cows

 It is a crucial enzyme responsible for effectively catalysing the reversible

hydration of CO2 in blood

 This enzyme has been found in virtually all living system, however only
members of alpha-CA gene family are found in mammals

 The active site of most carbonic anhydrase contains a zinc ion

 It is located in red blood cells, pancreatic cells, gastric mucosa, renal tubules
 Structural features
 In all isoenzymes , there exists one Zn(II) site per molecule.

There are at least five distinct CA families ( alpha, beta, gamma, delta and
zeta). These families have no significant amino acid sequence similarity .The
alpha CA s are found in humans

 In terms of activity various isoenzymes of alpha CA are classified as :

a)High activity forms (kcat =106 s-1) are labeled as CA II
b)Low activity forms (kcat=105 s-1) are labeled as CA I
c)Very low activity forms (kcat =103 s-1) are known as CA III

These are single chain polypeptides

In human CA II , Zn(II) is in a distorted tetrahedral geometry and it is
coordinated with three histidine–N sites (His-94,His-96,His-119) and the
fourth coordination site is occupied by a H2O molecule or a OH- ion

If source is human ,they are described as HCA I ,HCA II (H indicates human

source)
 Fig -Ribbon diagram of human carbonic anhydrase II,
with zinc ion visible in the Centre

Orange : enzyme backbone

grey sphere : Zn2+
Magenta : 3 His bound to Zn2+ and other hydrophillic side chains
Green : Hydrophobic amino acids nearby
Red : H2O
 Biological function of CA
 CA is a crucial enzyme that operates in animal cells, plant cells and in the environment to
stabilize CO2 concentration. The important biological functions are as follows

1. pH Regulation through hydration of CO2 and dehydration of

H2CO3
One of the important function of this enzyme in animals is to interconvert CO2 and
bicarbonate to maintain acid-base balance and fluid balance in different parts of our body.

(k =10^-1 s-1)

( (k =104 mol-1 s-1)

( ( k= 10^6 s-1)
 From the rate constant , it is evident that the rate acceleration
by CA at physiological pH is tremendously high . This CA
catalysed equilibration reaction plays an important role as a
buffer in human blood

Each molecule of CA can hydrate about one million molecules of

CO2 per second at body temperature ( 37 degree centigrade).

In this way ,the production of bicarbonate ions and protons

regulates the pH to maintain the acid-base balance in blood and
other tissues .
2. Enhances CO2 transport /storage(respiration/ photosynthesis)

In red blood cells, this enzyme performs the important role of receiving CO2 from
tissues such as active muscles and releasing in the alveoli of the lungs

CA also helps to control fluid balance in different parts of the body i.e. eye ,
kidney. This enzyme also function in the formation of Hydrochloric acid by the stomach

Fig - Transpot of CO2 to lungs through

the blood stream
Characteristics of Enzymatic
Activity

1. Prosthetic group

In CA , the nonprotein part or prosthetic group is the Zn

metal ion. If Zn2+ is removed from CA by dialysis
against the strong chelating agent like phenanthroline, the
apoenzyme becomes completely inactive . The activity
can be restored by adding Zn2+ ion in 1:1 molar ratio to
the apoenzyme .
2. Formation of Zn(II)- OH to offer a
better nucleophile

OH- is a better nucleophile than H2O . In fact the

hydration of CO2 by OH - is much faster than the hydration by
H2O.

But at physiological pH ,availability of OH- is unlikely. The CA

enzyme (N3)ZnII-OH2 generates the metal bound OH group
through deprotonation
The lewis acidity of Zn (II) is considerably high due to presence of three
imidazole moieties . The Imidazole moiety , comes from histidine residues ,
act as pi acid ligand by accepting the metal d electrons into the vacant Π*
orbitals of Im through Π back bonding (d-Π*)
This pi back bonding enhances the lewis acidity of Zn (II) in CA. The
imidazole moiety present at the suitable position facilitates the H+ transfer
to generate the active ‘Zn(II)-OH’ centre.

Crucial step
Plausible catalytic cycle of CA
Step 1 : deprotonation

Step 2: Carboxylation
The anionic oxygen then performs a nucleophillic
attack on carbon
Step 3 : Formation of Ring Structure

Anionic oxygen forms

a bond with Zn2+ to
make a ring-like
resonance

Step 4 : Formation of Bicarbonate

Addition of water
displaces the
bicarbonate ion
 4. Inhibition of enzymatic activity - The enzymatic activity is
very often inhibited by different anions like I- ,SH- ,N3 - ,CN- ,CNO- etc. and
neutral substances like sulfonamides (RCO2NH2) and imidazole. Probably
these inhibitors are positioned in a pocket close to the Zn(II) site . This
attachment blocks the active site of CA

Medical Application
Treat Glaucoma
 Carbonic anhydrase inhibitors, Sulfonamides are used in the treatment of
glaucoma to reduce the intraocular pressure (IOL) that has build up inside the
eye by 40% to 60% through blocking the enzyme action.

 Modified carbonic anhydrase enzymes have been used to replace methyl

diethanolamine ("MDEA") in carbon dioxide capture
 Zinc ion facilitates the H2O to create a H+ and nucleophillic
OH- .The nucleophillic OH- then attack the CO2 to convert it
into bicarbonate. Because of its high lewis acidic character
Zn ion can attract oxygen of H2O to facilitate the
deprotonation and makes easier for water to turns into a OH-
which is a better nucleophile.
 Reference
 Bioinorganic Chemistry by A.K Das
Elements of Bioinorganic Chemistry by G.N.
Mukherjee
A. S. GHAZAXFAR et. al. J. Biol. Chem. 1964,239,1065
T.H. MAREN Physiol. Rev.1967,47,4
William S. Sly et. al. Annu. Rev. Biochem. 1995,
64,375