**Carbonic Anhydrase and Heavy Metals**

Maria Giulia Lionetto, Roberto Caricato, Maria Elena Giordano, Elisa Erroi and Trifone Schettino *University of Salento - Dept. of Biological and Environmental Sciences and Technologies Italy* 

#### **1. Introduction**

Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc metalloenzyme catalysing the reversible hydration of CO2 to produce H+ and HCO3−. Its activity is virtually ubiquitous in nature. The fundamental role of this biochemical reaction in diverse biological systems has driven the evolution of several distinct and unrelated families of CAs. Five CA families, referred as α-, β-, γ-CA, δ, and ζ-CAs have been identified in animals, plants and bacteria (Hewett-Emmett and Tashian, 1996; Supuran, 2010). These are the α-CAs, present in vertebrates, bacteria, algae and plants; the β-CAs, predominantly in bacteria, algae and plants; the γ-CAs, mainly present in archaea and some bacteria; the δ-CAs and ζ-CAs only found in some marine diatoms (Supuran, 2010).

The monomeric α-carbonic anhydrases are by far the best studied, being found in animals. In mammals at least 16 different CA isoforms were isolated and several novel isozymes have also been identified in non-mammalian vertebrates. The α-CA isoenzymes differ in their kinetic properties, their tissue distribution and subcellular localization, and their susceptibility to various inhibitors. In general, there are three distinct groups of CA isozymes within the α-CA gene family. One of these groups contains the cytoplasmic CAs, which includes mammalian CA I, II, III, V, VII and XIII. These isozymes are found in the cytoplasm of various tissues, with the exception of the mitochondrial confined CA V. Another group of isozymes, termed the membrane-bound CAs, consists of mammalian CA IV, IX, XII, XIV and XV (Esbaugh and Tufts, 2006). These isozymes are associated with the plasma membranes of many different tissue types. The final group contains several very intriguing isozymes, CA VIII, X and XI, which are termed the CA-related proteins (CA-RP; Tashian et al., 2000). These isozymes have lost classical CA activity – the hydration/dehydration of CO2 – and have no known physiological function; however, their highly conserved nature does suggest a very important role in vertebrates (Tashian et al., 2000).

The β-carbonic anhydrases are dimers, tetramers, or octamers and include the majority of the higher plant CA isoforms (Kimber and Pai, 2000). The γ-carbonic anhydrase is a homotrimer that has been reported for the bacterium *Methanosarcina thermophila* (Alber and Ferry, 1994). The δ class has its prototype in the monomeric CA TWCA1 from the marine diatom *Thalassiosira weissflogii* (Roberts et al., 1997; Tripp et al., 2001). The ζ-CAs are probably monomer with three slightly different active sites on the same protein backbone (Xu et al 2008).

All CAs are metalloenzymes but whereas α-, β-, and δ-CAs use Zn(II) ions at the active site, the γ-CAs are probably Fe(II) enzymes (Ferry et al., 2010), but they are active also with bound Zn(II) or Co(II) ions, and the ζ-class uses also Cd(II) to perform the physiologic reaction catalysis (Lane et al., 2000; Lane et al., 2005).

CA plays key roles in a wide variety of physiological processes involving CO2 and HCO3 - . In animals the various CA isozymes are found in many different tissues and are involved in a number of different physiological processes, including bone resorption, calcification, ion transport, acid–base transport, and a number of different metabolic processes such as biosynthetic reactions (gluconeogenesis, lipogenesis, and ureagenesis). In algae and plants they play an important role in photosynthesis (Ivanov et al, 2007; Zhang et al., 2010; Cannon Gordon et al., 2010).

Considerable advances towards a detailed understanding of the catalytic mechanism of the zinc enzyme carbonic anhydrase have been made during the past years as a result of the application of crystallographic and kinetic methods to wild-type and mutant enzymes. Moreover, a great amount of work has been performed on CA inhibitors, first of all sulfonamides, RSO2NH2, which represent the classical CA inhibitors (CAIs) and are in clinical use for more than 50 years as diuretics and systemically acting antiglaucoma drugs (Supuran, 2010).

The review focuses on one interesting but less investigated aspect of the biochemistry of this metalloenzyme, encompassing several areas of interest from human health to environmental science: the relationships between carbonic anhydrase and heavy metals. Heavy metals are chemical elements with a density higher than 5.0 g/cm3, characterized by high reactivity, redox behaviour, and complex formation based on the characteristic of the outer *d* electron shell. In the scientific literature the following elements are normally ascribed to the heavy metal groups: aluminium, iron, silver, barium, beryllium, manganese, mercury, molybdenum, nickel, lead, copper, tin, titanium, tallium, vanadium, zinc. Some metalloids, such as arsenic, bismuthum, and selenium, are also included in the heavy metals groups.

Heavy metals generally regarded as essential for animals in trace amounts include zinc, the known cofactor of CAs, iron, copper, manganese, chromium, molybdenum and selenium. They are essential because they form an integral part of one or more enzymes involved in a metabolic or biochemical process. Besides essential metals, a number of other heavy metals, such as arsenic, lead, cadmium, mercury, have no known function in the body and are referred as toxic metals. However, also essential metals become toxic when their levels in the body exceed the homeostatic capacity of the organism. The intracellular levels of essential metals are regulated by transporters (which translocate metal across the plasma membrane) as well as by metallothionein and other metal binding proteins (Maret and Wolfgang, 2011). The toxicity of heavy metals is generally ascribed to their high affinity for nucleophilic groups like sulfhydryls. In fact they are soft donors and will therefore readily bind to soft acceptors such as sulphydryl groups.

Recently, a number body of evidence has emerged regarding the effect of several heavy metals on carbonic anhydrase catalytic activity and protein expression. These studies encompass a wide area of interest from human health to environmental sciences.

All CAs are metalloenzymes but whereas α-, β-, and δ-CAs use Zn(II) ions at the active site, the γ-CAs are probably Fe(II) enzymes (Ferry et al., 2010), but they are active also with bound Zn(II) or Co(II) ions, and the ζ-class uses also Cd(II) to perform the physiologic

CA plays key roles in a wide variety of physiological processes involving CO2 and HCO3

animals the various CA isozymes are found in many different tissues and are involved in a number of different physiological processes, including bone resorption, calcification, ion transport, acid–base transport, and a number of different metabolic processes such as biosynthetic reactions (gluconeogenesis, lipogenesis, and ureagenesis). In algae and plants they play an important role in photosynthesis (Ivanov et al, 2007; Zhang et al., 2010; Cannon

Considerable advances towards a detailed understanding of the catalytic mechanism of the zinc enzyme carbonic anhydrase have been made during the past years as a result of the application of crystallographic and kinetic methods to wild-type and mutant enzymes. Moreover, a great amount of work has been performed on CA inhibitors, first of all sulfonamides, RSO2NH2, which represent the classical CA inhibitors (CAIs) and are in clinical use for more than 50 years as diuretics and systemically acting antiglaucoma drugs

The review focuses on one interesting but less investigated aspect of the biochemistry of this metalloenzyme, encompassing several areas of interest from human health to environmental science: the relationships between carbonic anhydrase and heavy metals. Heavy metals are chemical elements with a density higher than 5.0 g/cm3, characterized by high reactivity, redox behaviour, and complex formation based on the characteristic of the outer *d* electron shell. In the scientific literature the following elements are normally ascribed to the heavy metal groups: aluminium, iron, silver, barium, beryllium, manganese, mercury, molybdenum, nickel, lead, copper, tin, titanium, tallium, vanadium, zinc. Some metalloids, such as arsenic, bismuthum, and selenium, are also included in the

Heavy metals generally regarded as essential for animals in trace amounts include zinc, the known cofactor of CAs, iron, copper, manganese, chromium, molybdenum and selenium. They are essential because they form an integral part of one or more enzymes involved in a metabolic or biochemical process. Besides essential metals, a number of other heavy metals, such as arsenic, lead, cadmium, mercury, have no known function in the body and are referred as toxic metals. However, also essential metals become toxic when their levels in the body exceed the homeostatic capacity of the organism. The intracellular levels of essential metals are regulated by transporters (which translocate metal across the plasma membrane) as well as by metallothionein and other metal binding proteins (Maret and Wolfgang, 2011). The toxicity of heavy metals is generally ascribed to their high affinity for nucleophilic groups like sulfhydryls. In fact they are soft donors and will therefore readily

Recently, a number body of evidence has emerged regarding the effect of several heavy metals on carbonic anhydrase catalytic activity and protein expression. These studies

encompass a wide area of interest from human health to environmental sciences.


reaction catalysis (Lane et al., 2000; Lane et al., 2005).

Gordon et al., 2010).

(Supuran, 2010).

heavy metals groups.

bind to soft acceptors such as sulphydryl groups.
