Introduction of Carbonic Anhydrases
The carbonic anhydrases (CAs, EC 18.104.22.168) are a kind of zinc enzymes, widely distributed in prokaryotes and eukaryotes, and are encoded by seven different gene families which is unrelated in evolution. In solution, these enzymes catalyze the exchange between CO2 and HCO3-. Although these reactions can conduct without CA, the reaction rate is fairly slow. Researchers generally think that CA is needed in many metabolic pathways in organisms to make sure a quick conversion between CO2 and HCO3-.
The most crucial functions of these enzymes in animals is to balance the acid-base in blood and other tissues, and to assistant transport CO2 out of tissues.
Reaction and mechanism
CO2 is the final product of the respiration of all living things, from microorganisms to mammals. Actually, the sugars produced from sunlight and CO2 by plants and microorganisms are vital for every life on earth, lives can use then energy from the metabolic breakdown of the sugars to move, grow and breed. The interconversion of CO2 and HCO3- is spontaneously balanced to maintain the equilibrium between dissolved inorganic CO2, H2CO3, HCO3- and CO32- (Figure 1). This dynamic procedure is catalyzed by CAs and involves in relevant reactions of all lives.
Figure 1. CAs catalyze the reaction of the reversible hydration of CO2.
The necessary part for α-CAs' catalytic activity is the metal ion. According to X-ray crystallographic results, there is a metal ion at the bottom of a 15 Å deep active site cleft (Figure 2). It is coordinated by 3 histidine residues and a water molecule/hydroxide ion. There is also an engagement between zinc-bound water and the hydroxyl moiety of Thr199 as a hydrogen bond interactions, and this is a bridge to the carboxylate moiety of Glu106. In this manner, the nucleophilicity of the zinc-bound water molecule is enhanced and the CO2 will be directed to a site where is better for the nucleophilic attack.
Figure 2. The Zn(II) ion coordination in the hCA II active site, with the His94, His96 and His119 histidine ligands and the gate-keeping residues (Thr199 and Glu106) shown.
Classification of Carbonic Anhydrases
The various CA proteins grouped into different families are named by Greek letters and present in an order of their discovery. They are the α-CA first found in vertebrates, β-CAs in Chlamydomonas reinhardtii and in terrestrial plants, γ-CA, δ-CA, ζ-CA, η-CA and θ-CA which are the two latest discovered CA families. Indeed, there is barely relations between each protein family in phylogenetic, which means little or no similarities can be founded on their sequences or structures when comparing the α- and γ-CA classes. Besides, the active sites on CA for coordinating with the Zn ions are not conserved too. But, researchers also find that there are some similarities in the active site of some CAs. For example, the active sites on ζ- and θ-CAs are structurally analogous to β-CAs while the active sites on δ-CAs are similar to α-CAs.
Structure and function
The α-CAs are widely distributed in plants, green algae, diatoms, cyanobacteria and animals and are involved in many reactions (Figure 3). Their structures and amino acid sequences are quite different from other CA families. There is a main structure as a central β-sheet in α-CAs, it consists of 10 β-strands with a surrounding of 7 peripheral α-helices. The active site of the α-CA is in the central β-sheet and this structure can coordinate the zinc atom with 3 histidine residues and a water molecule. Although people used to considering that α-CA is the only CA family without multimers, recently some researchers find there are some α-CA monomers in a form of dimerization.
Figure 3. Reactions catalyzed by α-CAs. Ar = 2, 4-dinitrophenyl, R = Me; Ph.
β-CAs and ε-CAs
The first report about the existence of CAs in plants was reported in 1936. Researchers then underwent protein sequence analysis, finding that those CAs from chloroplast differ from the α-CAs, these new CAs are now known as β-CAs. The β-CAs are widely found in plants, algae, cyanobacteria, and non-photosynthetic bacteria but not from animals. The basic structure of β-CA monomer is also different from α-CAs. There is a central β-sheet which is comprised of only 4 parallel β-strands and is surrounded by α-helices. After oligomerizing to dimers, β-CA offers 2 active sites which contain a histidine residue, a water molecule and a zinc atom coordinated by two cysteine residues. These dimers then further interact to form tetramers and octamers. The ε-CAs which are recognized as a highly modified β-CA are found in cyanobacteria. ε-CAs not only function as an enzyme, but also take part in forming the carboxysome shell in cyanobacteria.
γ-CAs were first found in the archaeon Methanosarcina thermophila. Then researchers also discovered γ-CAs in plants and photosynthetic bacteria but not reported in animals. In archaebacteria there is a zinc active site coordinated by three histidine residues and one water molecule. The three active sites of the γ-CAs are provided by two monomers. One provides 2 zinc-coordinating histidine residues and another one providing the third zinc-coordinating histidine residue. There is also a modified γ-CA in cyanobacterial namely CcmM protein. A γ-CA domain is on the N-terminal of CcmM and 3 to 4 RbcS domains are on the C-terminal. This structure allows CcmM to coordinate Rubisco packaging in the carboxysome. Although not all CcmM proteins have the CA activity, they are still crucial for carboxysome packaging. Indeed, if the CcmM protein in cyanobacteria is inactive, we can find a β-CA to convert HCO3- to CO2 for rubisco. γ-CAs are also found in the mitochondria of green algae and diatoms. But there is no CA activity found in eukaryotic algae or plants so far.
δ-CAs and ζ-CAs
Till today, δ-CAs (e.g. TWCA1) and ζ-CAs (e.g. CDCA1) have only been found in diatoms and coccoliths. According to the report in 2012, there is a Zn-binding region of CDCA1, which is repeated three times. What makes CDCA1 different from other CAs is that CDCA1 can interact with other metals like Cd besides Zn. In fact, the Zn level is low in oceanic environments, plants need to adapt and evolve to have the ability to bind other metals.
η-CAs was first reported in 2015. Although there is a report that the Zn coordination pattern of η-CAs is a little close to that of α-CAs, η-CAs’ detailed structure is still not clear.
θ-CAs are the most recent family of CAs reported. Pt43233, one of the θ-CAs found in diatom, is located to the thylakoid lumen. It can catalyze the HCO3- pool to form CO2 in the diatom chloroplast. The pH of the thylakoid lumen is below the pKa of the conversion of CO2 to HCO3-, Pt43233 would prefer to produce CO2 from any HCO3- transported into the thylakoid. Diatoms will grow slowly on air levels of CO2 if Pt43233 is absent and will show a lower affinity for inorganic carbon. LCIB/LCIC complex of C. reinhardtii is another member of θ-CAs. It has been well-studied that the LCIB gene encodes a θ-CA which is a chloroplast stromal protein surrounding the chloroplast pyrenoid. Absent of LCIB makes C. reinhardtii to need a high CO2 concentration for growth and photosynthesis. LCIB/LCIC can recapture the CO2 leaking out of the pyrenoid then reform it as the HCO3- back into the pyrenoid.
Industrial Applications and Inhibitors
As the second-leading cause of death in the world, infectious diseases and their drug development are well studied but the antibiotic-resistant microorganisms are also widely found. These antibiotics usually targets to inhibit the following biological processes: cell wall biosynthesis, protein biosynthesis, DNA and RNA biosynthesis, and folate biosynthesis. Because CAs are important for pathogenic microorganisms life cycle and their inhibition would result in growth impairment and defects, researchers now focus on searching for new anti-infectives in inhibiting CAs function.
By substituting the non-protein zinc ligand or by adding to the metal coordination sphere generating trigonal-bipyramidal species, there are main classes of CAs inhibitors (CAIs) as follows: the metal complexing anions, the unsubstituted sulfonamides and the dithiocarbamates. Prontosil is the first sulfonamide with effective anti-bacterial activity and it is a prodrug, in another word the real antibacterial agent is sulfanilamide which is generated by the in vivo reduction of prontosil. After the finding of sulfanilamide as a bacteriostatic agent, a number of analogs has been widely used in clinical, such as the clinically used derivatives acetazolamide (AAZ), methazolamide (MZA), ethoxzolamide (EZA), dichlorophenamide, dorzolamide (DZA) and brinzolamide (BRZ). Anions also can bind to the CAs, but the efficiency is less than the sulfonamides. By studying the anion inhibitors, we will have a better understanding the inhibition/catalytic mechanisms of these enzymes fundamental for many physiologic processes, which in turns helps us to design new types of inhibitors with clinical benefits to manage a variety of CA involved diseases. Another kind of CAIs is the dithiocarbamates (DTCs). DTCs will coordinate through one sulfur atom to the Zn (II) ion at the enzyme active site. Besides, they can also interact with the conserved Thr199 amino acid residue.