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

Abstract

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...

Structural Mechanics of the pH-dependent Activity of β-Carbonic Anhydrase from Mycobacterium tuberculosis

“…The roles of the equivalents of Asp-53 and Arg-55 have been analyzed by site-directed mutagenesis of the M. thermoautotrophicum enzyme (47). The Asp 3 Ala mutation reduced the k cat /K m but not to an extent that would suggest that the residue plays an essential role in the reaction.…”

Evolution of carbonic anhydrases in fungi

“…In addition to these three residues, an aspartic acid and an arginine are also structurally conserved in every known -CA sequence (Tripp et al 2001). These two residues presumably assist in a number of catalytic steps, including substrate binding, proton shuZing and product release, or act as the fourth zinc ligand (Mitsuhashi et al 2000;Cronk et al 2001;Smith et al 2002). These Wve amino acid residues are completely conserved among the 82 fungal -CAs (Supplementary Figure 1).…”

Structure and Function of Carbonic Anhydrases from Mycobacterium tuberculosis

“…The authors propose that this residue functions as a base that activates a water molecule and that the resulting hydroxide displaces the aspartyl group as the fourth zinc ligand in the next steps of the reaction. The roles of this aspartyl side chain and the neighboring arginine residue (the equivalents of Asp 37 and Arg 39 in Rv1284, both of which are conserved in the whole family) have been analyzed by site-directed mutagenesis for the M. thermoautotrophicum enzyme (34). The Asp3 Ala change reduced the k cat /K m value, but not to an extent that would suggest a vital role for the residue in the reaction mechanism.…”