CARBONIC ANHYDRASE: MECHANISM,

STRUCTURE AND IMPORTANCE IN

HIGHER PLANTS

SUBMITTED TO: DR. ROHEENA

SUBMITTED BY:
FARIHA PERVAIZ 12
FARWA AMJAD 13
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INTRODUCTION
Carbonic anhydrase is a ubiquitous zinc-metalloenzyme important for photosynthesis
because of its property to convert CO2 to HCO3- reversibly. The occurrence of carbonic
anhydrase in plants was confirmed by Bradfield in 1947. The rate of photosynthesis and
CO2 fixation gets directly affected due to any change in the activity of carbonic anhydrase.
It is the only carbon metabolism enzyme which shows fluctuations in the activity among
different species with varying CO2 concentration in the environment. Carbonic anhydrase
is one of the fastest enzymes known for hydrating 106 molecules of CO2 per second. The
rate of reaction of this enzyme is typically limited by the rate of diffusion of its substrates.
In leaves of C3 plants the enzyme represents 1% to 2% concentration of the total soluble
protein, second only to RuBisCO in concentration. About 86% to 95% of the total carbonic
anhydrase is found in chloroplasts, while the rest is completely restricted to the cytosol of
mesophyll cells. A correlation was found by Khan between carbonic anhydrase activity
and photosynthetic rate suggesting that the enzyme serves as a biochemical marker for
productivity as this helps in carbon sequestration. The activity of Carbonic anhydrase is
mainly keeping up by light, Zn and CO2 concentration. This enzyme provides the supply
of CO2 to the phosphoenolpyruvate carboxylase in C4 and Crassulacean Acid Metabolism
(CAM) plants and RuBisCO in Calvin-Benson cycle (C3 plants) (Figure 1). Carbonic
anhydrase is concerned in a mixture of physiological processes. The present review,
however, lays emphasis on recent aspects of carbonic anhydrase in higher plants.

Mechanism of action of carbonic anhydrase

The carbonic anhydrase active sites contain a zinc ion and all share a similar catalytic
activity. In the carbonic anhydrase enzyme, a zinc prosthetic group is coordinated in three
positions by histidine side-chains. The active site of the enzyme “E” contains a specific
pocket for CO2 which brings it closer to the hydroxide group attached to the zinc.
Therefore, this causes the electron-rich hydroxide ion to attack the CO2 thereby creating a
bicarbonate molecule.

Structure
Carbonic Anhydrase structures are unique and are evolutionary characterized into
dissimilar classes, such as alpha anhydrases (α), beta anhydrases (β), gamma anhydrases
(γ) and delta anhydrases (δ). These are found majorly, whereas, there also exist two more
class of anhydrases called epsilon anhydrases (ε) and zeta anhydrases (ζ), which are limited
to the bacterial kingdom and protists, respectively. The α-class is found throughout the
animal kingdom, in the periplasm. The ɑ-carbonic anhydrases were widely identified in
vertebrates, algae and in eubacteria as reported by Chirica et al. The β-carbonic ahydrases
are mostly found in higher plants; micro-algae; Eubacteria; Archaebacteria; Cyanobacteria
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and Fungi. The γ-carbonic anhydrases are discovered in the algae. δ-carbonic anhydrases
have only been described in some diatoms. The δ-carbonic anhydrase families appears to
be a case of convergent evolution with almost no sequence similarity with the ɑ-, β- or γ-
carbonic anhydrase types. ε-carbonic anhydrase is part of the carboxysome shell and has
additional domains that serve the function in bacteria. ζ-carbonic anhydrase is limited to
maline protists and resembles the β-carbonic anhydrase family, with other metals such as
Cd or Co substitute for Zn.

Figure 1: A generic model for the operation of a CCM showing the potential roles of CA
in the process

Isozymes
Isozymes or isoenzymes are basically enzymes that only differ in the sequences of amino
acid but catalyze the same chemical reaction. Carbonic anhydrase has various isozymes
that play a great role in various physiological processes of higher plants.

Zinc as a cofactor of beta (β) carbonic anydrase

A co-factor is a non- proteinacious chemical compound or metallic ion that is required for
assisting a smooth biochemical transformations and biological activities. “Carbonic
anhydrase a metalloenzyme has property to convert CO2 to HCO3- reversibly”. The Zn acts
as a cofactor in carbonic anhydrase and plays an important role in carboxylation. The
presence of Zn ion (Zn2+) on active site which binds with the enzyme and causes reduction
in pKa value and finally, allows the nucleophilic attack on CO2.
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For the activation of catalytic site, water bounds to Zn, which can be ionized to form a
hydroxide bridge (carbonic anhydrase), polarized by a base to generate nucleophilic
catalysis or displaced by the substrate (Figure 2).

Different gene family of carbonic anydrases in higher plants

Higher plants contain various carbonic anhydrases which shows variations in their
distribution in terms of the number of genes present in each family. For example,
Arabidopsis has 19 carbonic anhydrase genes (8ɑ-CA, 6β-CA, 5γCA) [10], rice also have
a similar number of genes present in them. The alpha-carbonic anhydrase (ɑ-CA), beta-
carbonic anhydrase (β-CA) and gamma-carbonic anhydrase (γ-CA) isoforms share the
same general catalytic mechanism in higher plants, despite their structural difference.

Enzymatic characterization of carbonic anhydrases in higher plants

Primary protein structures of the various carbonic anhydrases have distinct differences, but
their secondary and tertiary structures must be fairly different. Carbonic anhydrases show
a prominent similarity in their metal-coordinating sites. As carbonic anhydrase is a
metalloenzyme, it requires Zn2+ for its activity. But some carbonic anhydrases require
cobalt (Co) and cadmium (Cd) for their activity as reported by Morel et al.
The activity of carbonic anhydrase varies in different plants. In tobacco the activity of
carbonic anhydrase enzyme follows pattern: leaves>stem>pods, also the enzyme has been
found in plant roots and fruits. Diverse compartmentalization among organs, tissues, and
cellular organelles is being exhibited by Carbonic anhydrase with different physiological
roles. Chloroplastic carbonic anhydrase contents in higher plants are saturated while the
cytoplasmic carbonic anhydrase contents are inadequate.

Figure 2: Simplified configuration of zinc reactions with water

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Higher plants show similarity between cytoplasmic and chloroplastic carbonic anhydrases
in kinetic properties, affinity for CO2 and sensitivity to inhibitors. It has been shown that
from the purification of the Solanum cytosolic isoform that it is structurally and
biochemically similar to the chloroplastic form, although its monomeric mass is larger. The
difference of expression between cytoplasm and chloroplast carbonic anhydrase because
difference of promoter region between the two kinds of carbonic anhydrases. The
bicarbonate (HCO3–) pools are maintained by cytoplastic carbonic anhydrases, and
compensate leakage of free CO2 from the cytoplasm. Chloroplast carbonic anhydrases
appear to be associated with other enzymes of the Calvin cycle in a large multienzyme
complex. Chloroplast fraction in C3 is associate with the carbonic anhydrase activity, there
are at least 10 to 15% of the total activity is cytoplastic.

Importance of carbonic anhydrase

Carbonic anhydrases (CAs; EC 4.2.1.1) is a metalloenzymes with zinc ligands that catalyze
the reversible hydration of carbon dioxide to bicarbonate. Among the most efficient
enzyme, carbonic anhydrases acts over a broad spectrum of pH. Knowledge of carbonic
anhydrase activity in plants has expanded significantly since the first scientific report was
published 78 years ago. During this time majority of studies have focused on the key role
of carbonic anhydrases in inorganic carbon fixation, respiration and CO2 transport and
electrolyte secretion among other things and it was found that carbonic anhydrases helps
in the regulation of chloroplast pH and protect stroma enzymes against denaturation during
rapid and drastic changes in light conditions. Betacarbonic anhydrase involvement in the
CO2 concentrating mechanism (CCM), which increases the content of this gas in close
proximity to RuBisCO and consequently decreases photorespiration.
 Carbonic anhydrase plays an important role in the mitigation of climate change in
response to increasing concentration of CO2 in the atmosphere.
 So far, a multiplicity of carbonic anhydrase isoforms in Arabidopsis has been found,
with eight αCA (AtaCA1-8) and six βCA genes (AtbCA1-6). The physiological role
of alpha-carbonic anhydrase (AtαCA1) was widely expressed in the aboveground
tissues; it has been shown to be localized to the chloroplast stroma following
transport through the secretory pathway and N-glycosylation. Gama- carbonic
anhydrase (γ-CAs) play a biological role in mitochondrial physiology, including
experimentally proven the importance of gama- carbonic anhydrases for complex I
assembly. Moreover, γ-CAs is implicated in male sterility, plant growth and
embryogenesis.
 Molecular, biochemical and genetic studies of carbonic anhydrases analyzed in
various tissues of many organs and plant species suggested an effect of carbonic
anhydrase on a wide range of diverse biological processes, including pH regulation,
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gas and ion exchange, the provision of bicarbonate for anaplerotic reactions, and
fatty acid biosynthesis.
 The increased amount of two carbonic anhydrases identified with MALDI-TOF MS
could be involved in freezing tolerance during cold acclimation in the purified
plasma membrane fraction from Arabidopsis leaves.
 There is evidence that during the immune response of plants to pathogens, carbonic
anhydrase plays a very important role, acting as a salicylic acid binding protein3
(SABP3). Experimentally, this dual activity of chloroplastic carbonic anhydrases
has been observed in tobacco. Finally, the carbonic anhydrase function in plant
immunity has been defined enigmatically as oxidative stress protection.

Role of beta (β) carbonic anhydrase in photosynthesis

Plants need various enzymes for carrying out photosynthesis. The rate of photosynthesis
and CO2 fixation (under limited CO2 condition) gets directly affected due to any change in
activity of carbonic anhydrase. It is the only carbon metabolism enzyme which shows
fluctuations in the activity among different species with varying CO2 concentration in the
environment. Its activity gets to decrease rapidly into the air bubbles at low CO2 emissions
while its activity increases at high CO2 emissions. This has shown a strong co-relation
between the activity of carbonic anhydrase and CO2 concentration, and therefore, this
enzyme is critical for carrying the process of photosynthesis.

Functions of beta (β) carbonic anhydrase

The functions of Carbonic anhydrase in photosynthetic CO2 fixation are:
1. Beta (β) carbonic anhydrase helps In C3 plants to raise the concentration CO2 in the
chloroplast, which plays a significant role as a substrate by the RuBisCO enzyme,
therefore increasing its carbon fixation (carboxylation) rate and hence participates
in the reaction by incorporating CO2 into carbohydrates during photosynthesis
where CO2 can be used only as Carbon and not the carbonic acid or the bicarbonate
ion.
2. Similarly, in C4 plants carbonic anhydrase associates with phosphoenolpyruvate
(PEP) carboxylase (PEPC) provides continuous supply of HCO3- at the site of
carbon fixation. CO2 hydration to form HCO3- is used as a substrate by
phosphoenolpyruvate carboxylase (PEPC) in C4 and CAM plants. This is supported
by the fact that carbonic anhydrase of C4 leaves is entirely confined to the cytosol
of mesophyll cells while bundle sheath cells contain very low or no carbonic
anhydrase activity. Thus, carbonic anhydrase in mesophyll cells rapidly converts
diffusing atmospheric CO2 to HCO3- at rates that are compatible with those for
photosynthesis. In C4 plants (Figure 3), Carbonic anhydrase catalyzes the first
critical step of C4 photosynthesis, the hydration of CO2 to bicarbonate, which PEPC
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uses as the substrate for carboxylation of PEP to oxaloacetate in the cytosol of

mesophyll cells. The inorganic carbon substrate HCO3– of this enzyme was
recognized as being supplied by carbonic anhydrase.
3. The carbonic anhydrase helps in diffusion of CO2 through the plasma membrane
and the chloroplast.
4. There are various carboxylation enzymes which help in photosynthesis but among
all Carbonic anhydrase plays a terrific role in photosynthesis because it has property
to convert reversibly CO2 to HCO3-. Due to participation of active CO2 transport
through the plasma of the membrane by converting CO2 into HCO3- is how Carbon
enters the cell. Due to the increase in activity of this enzyme, the rate of fixation of
carbon in plants also increases. Carbonic anhydrase holds a special significance for
global forests because it sequesters about 90% of terrestrial carbon pool, which can
lower the impact of growing global warming. The above functions make carbonic
anhydrase a biochemical marker for carbon sequestration which helps in the
mitigation of global warming.

Role of beta (β) carbonic anhydrase in stomatal closure

The stomata are a minute opening or pore through which exchange of gases takes place.
They are mostly found on under-surface of plant leaves. The enzyme carbonic anhydrase
plays a great role in stomatal closure as explained below:

Figure 3: Simplified diagram of the C4 and C3 reactions

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Figure 4: Simplified diagram of the central reaction for stomatal closure

During the dark phase of photosynthesis, the amount of CO2 fixation gets depleted while
the process of respiration continues which leads to increase in CO2 level by shifting the
central reaction (Figure 4) towards the right which results in a decrease of pH. The
decreasing pH causes the inactivation of various enzymes such as amylase, which stops the
mechanism of hydrolysis of starch and glucose, resulting in a decrease of the osmotic
gradient in occlusive cells causing loss of water and closure of ostiole.

CONCLUSION
Carbonic anhydrase plays critical role in all photosynthetic CO2 concentrating
mechanisms. Carbon dioxide being main culprit behind the global warming is cause of
concern for present generation as well as for the future generations. Therefore, need of hour
is to think of the present and sustaining of the future. This review attempts to present a
picture of the physiological role of carbonic anhydrase in plants, in the fixation of
greenhouse gas like CO2 from the atmosphere and its role as biochemical marker for carbon
sequestration.
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