A Structure−Function Study of a Proton Transport Pathway in the γ-Class Carbonic Anhydrase from <i>Methanosarcina thermophila</i>

Tripp, Brian C.; Ferry, James G.

doi:10.1021/bi0001877

Cited by 69 publications

(88 citation statements)

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“…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 intermolecular proton transfer to an accepting buffer molecule in the surrounding media (Equation 5). Kinetic analyses of site-specific replacement variants of Cam have identified essential residues (11,12). All of the biochemically characterized CAs from the ␣, ␤, and ␥ classes are categorically described as zinc metalloenzymes; however, there are few reports investigating the role of metals other than zinc.…”

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A Role for Iron in an Ancient Carbonic Anhydrase

Tripp

Bell

Cruz

et al. 2004

Journal of Biological Chemistry

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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...

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A Role for Iron in an Ancient Carbonic Anhydrase

Tripp

Bell

Cruz

et al. 2004

Journal of Biological Chemistry

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141

123

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“…Since CA inhibitors have been shown to reduce IOP exclusively by lowering the aqueous humour flow and these compounds have been used for the treatment of glaucoma for years 11,12 . The rate-determining step for the CO 2 hydration reaction catalyzed by CAs is the proton transfer reaction from the water bound to the Zn(II) ion to the reaction medium, with generation of the zinc hydroxide species of the enzyme [13][14][15][16][17][18][19][20] . Several anions behave as inhibitors for the CO 2 hydration reaction 21,22 and coordinate directly to Zn(II) ion.…”

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Synthesis and characterization of phenolic Mannich bases and effects of these compounds on human carbonic anhydrase isozymes I and II

Büyükkıdan

Bülbül

et al. 2012

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…In the ␣-class HCAII, the proton first undergoes intramolecular transfer to the proton shuttle residue His-64 (40). In the ␥-class enzyme Cam, this proton transfer is to Glu-84 (37). The proton is subsequently transferred to an accepting buffer molecule in the surrounding media during an intermolecular proton transfer step.…”

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Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

2002

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The roles of an aspartate and an arginine, which are completely conserved in the active sites of ␤-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the ␤-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k cat /K m value depends only on the rate constants for the CO 2 hydration step, whereas k cat also depends on rate constants from the proton transfer step ( Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide:This enzyme, which is present in species from all three domains of life, plays a critical role in many diverse physiological processes such as respiration, photosynthesis, and CO 2 fixation. Based on amino acid sequence comparisons, carbonic anhydrases belong to four genetically distinct classes (␣, ␤, ␥, and ␦) of independent origins (38). The crystal structures for representatives of the ␣, ␤, and ␥ classes have now been determined (3, 7, 9-13, 16, 18, 19, 23, 26, 35, 36). Although the structure of the recently identified ␦-class prototype from the diatom Thalassiosira weissflogii has not yet been solved, extended Xray absorption fine-structure analyses suggest that the activesite zinc is coordinated by three histidines and one water molecule, as found in the ␣ and ␥ classes (6). The structures of the ␤-class carbonic anhydrases (7,18,26,36) indicate striking differences between this class and the others. For example, the active-site zinc in the ␤-class enzymes is coordinated by two cysteines, one histidine, and one water molecule.The kinetic properties of the human ␣-class isozymes have been comprehensively investigated and follow a common zinchydroxide mechanism for catalysis (24, 30). The catalytically active group in this mechanistic model is the zinc-bound water, which ionizes to a metal-bound hydroxyl which attacks CO 2 . Despite considerable structural differences in the active sites of these enzymes, the catalytic mechanisms of both ␥-and ␤-class enzymes resemble those of human ␣-class HCA II (1,14,15,28,31). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps (where E ϭ enzyme and B ϭ buffer):The first step is the interconversion between carbon dioxide and bicarbonate (equations 2a and 2b) and involves the nucleophilic attack of the zinc-bound hydroxyl on the CO 2 molecule. In the ␣-class enzyme, the zinc-bound oxygen forms a hydrogen bond with 42,43). The role of this conserved gatekeeper residue is to prevent nonprotonated atoms from binding effectively. In addition, Thr-199 has been proposed to electrophilically activate the CO 2 molecule by forming a hydrogen bond with CO 2 through its backbone amide. The second step of the proposed mechanism is the regeneration of the zinc hydroxide at the active site of the enzyme (equations 2c and 2d), involving proton transfer events. The k cat /K m value depends only on the rate...

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A Structure−Function Study of a Proton Transport Pathway in the γ-Class Carbonic Anhydrase from Methanosarcina thermophila

Cited by 69 publications

References 51 publications

A Role for Iron in an Ancient Carbonic Anhydrase

A Role for Iron in an Ancient Carbonic Anhydrase

Synthesis and characterization of phenolic Mannich bases and effects of these compounds on human carbonic anhydrase isozymes I and II

Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

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