A Role for Iron in an Ancient Carbonic Anhydrase

Abstract

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (␣ class) and plants (␤ class); however, two additional classes (␥ and ␦) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo- Research in the intervening years has shown that CA is one of the most widely distributed enzymes in nature (2, 3) and continues to be intensely investigated. Amino acid sequence comparisons identify four classes (␣, ␤, ␥, and ␦) of independent origins (4). Isozymes of the ␣ class are found in virtually all mammalian tissues where they function in diverse essential processes. The ␤ class is ubiquitous in plants and algae, where it is indispensable for the acquisition and concentration of CO 2 for photosynthesis. CA plays a role in the sequestration of atmospheric CO 2 in carbonates, and the global cycles of silicon and carbon are linked by CA in diatoms (5); thus, CA plays an important role in major geochemical and atmospheric processes. Members of the ␤ and ␥ classes are wide spread in physiologically diverse procaryotes from both the Bacteria and Archaea domains. Indeed, the genome of Escherichia coli contains two ␥ class homologs and two ␤ class homologs (2).Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first CA to be characterized from either an anaerobic organism or the Archaea domain (6). Sequence analyses approximate the evolution of the ␥ class at the estimated time of the origin of life (2). The crystal structure of Cam purified aerobically from E. coli reveals a homotrimer with a subunit fold composed of a left-handed ␤-helix motif followed by short and long ␣-helix structures (7). Each of the three active sites contain three histidines that coordinate a zinc ion. Two of the metal-binding histidines are donated by one monomer, and the third histidine from an adjacent monomer. Other residues in the active site of Cam are also donated from adjacent monomer faces and bear no resemblance to residues in the active site of the well characterized ␣ class CAs for which specific functions have been assigned.Kinetic investigations of the ␣ class CAs reveal a "zinc hydroxide" mechanism for catalysis (8) that also extends to both the ␤ and ␥ classes (9, 10). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps,where E is enzyme, and B is buffer. The first step is the interconversion between CO 2 and bicarbonate (Equations 2 and 3) involving a nucleophilic attack of the zinc-bound hydroxyl on the CO 2 molecule. The second step is regeneration of the zinc-bound hydroxide, which involves intramolecular proton transfer from the zinc-bound water to a proton shuttle residue (Equation 4) and interm...

Inhibition of Carbonic Anhydrase II by Thioxolone: A Mechanistic and Structural Study

“…CAs use a two-step "ping-pong" mechanism (2-5) that typically involves a zinc ion cofactor functioning as a Lewis acid to ionize a water molecule, although other metal ion cofactors are known (6)(7)(8). CAs are found in most eukaryotic and many microbial organisms (9,10).…”

Carbonic Anhydrase and Heavy Metals

“…The different results obtained in aerobic and anaerobic conditions is explained by the fact that in aerobic conditions Fe 3+ is oxidized and rapidly loss from CAM enzyme, substituted by Zn 2+ contaminating buffers not treated with chelating agents. These results indicate Fe 2+ as the physiologically relevant metal [MacAuley et al, 2009;Tripp et al, 2004] in the active site for CAM enzyme. Interestingly, evidence for the role of ferrous ion in CA has been obtained also for the class.…”

Malaria parasite carbonic anhydrase: inhibition of aromatic/heterocyclic sulfonamides and its therapeutic potential

“…The large variations of activity were also observed for the coumarines 21-32, with only one compound 31 possessing submicromolar pfCA inhibitory activity, the vast majority of these sulfonamides being medium potency inhibitors. The standard CAI acetazolamide, AZA, behaves as a strong pfCA inhibitor too, with a KI of 0.32 毺 M. However, it is difficult to rationalize these data in the absence of an X-ray crystal structure of this enzyme, but we can state that small structural variations in the scaffold of our tested sulfonamides lead to very different inhibition profiles, which is noteworthy, meaning that it is possible to detect much more effective pfCA inhibitors by an intense screening effort of various libraries of structurally diverse compounds.…”

“…The type of 毩 -CAs kinetic mechanism reveals a zinc hydroxide catalysis that also extends to the 毬 -and 毭 -CA families [31,38] . This can be summarized as,…”

“…The CAs in protozoa and helminthes parasites are found sparsely [24][25][26][27][28] . Recent studies on the biochemistry and the crystal structure of CAs from various organisms reveal that they evolved independently al least five times, with five genetically distinct enzyme families known to date: the 毩 -, 毬 -, 毭 -, 毮 -, and 毱 -CAs [29][30][31][32] . These are the 毩 -CAs (mainly in mammals, vertebrates, some bacteria, algae, protozoa and cytoplasm of plant); the 毬 -CAs (ubiquitously in bacteria, algae and plant chloroplast, many fungi, some archaea); the 毭 -CAs (predominantly in archaea and some bacteria); whereas the 毮 -and the 毱 -CAs seem to be present only in marine diatoms) [19,33] .…”