Aspirin: A Suicide Inhibitor of Carbonic Anhydrase II

Abstract

Carbonic anhydrase II (CAII) is a metalloenzyme that catalyzes the reversible hydration/dehydration of CO₂/HCO₃^-. In addition, CAII is attributed to other catalytic reactions, including esterase activity. Aspirin (acetyl-salicylic acid), an everyday over-the-counter drug, has both ester and carboxylic acid moieties. Recently, compounds with a carboxylic acid group have been shown to inhibit CAII. Hence, we hypothesized that Aspirin could act as a substrate for esterase activity, and the product salicylic acid (SA), an inhibitor of CAII. Here, we present the crystal structure of CAII in complex with SA, a product of CAII crystals pre-soaked with Aspirin, to 1.35Å resolution. In addition, we provide kinetic data to support the observation that CAII converts Aspirin to its deacetylated form, SA. This data may also explain the short half-life of Aspirin, with CAII so abundant in blood, and that Aspirin could act as a suicide inhibitor of CAII.

Keywords: Aspirin; X-ray crystallography; carbonic anhydrase; esterase; salicylic acid.

Figure 1

Structure of carbonic anhydrase II (CAII) complexed with salicylic acid (SA). The hydrophobic face of CAII is shown as the orange surface while the hydrophilic face is shown as violet. Zinc is depicted as a magenta sphere with critical binding residues shown in sticks. Bound salicylic acid is shown in green sticks. Top insert, active site with SA interactions and hydrogen bonds shown in dashes; bottom insert, electron density for SA shown as blue mesh. PDB: 6UX1.

Figure 2

Inhibition curve of CAII with SA. Calculated IC50 of 6.6 mM. The error bars represent the standard deviation of 3 kinetic experiments performed.

Figure 3

Structure of Aspirin modeled into the active site of CAII. The hydrophobic face of CAII is shown as orange surface while the hydrophilic face is shown as violet. Zinc is depicted as a magenta sphere with critical residues shown in sticks. Bound Aspirin is shown in pink sticks. V134 and W204 are unlabeled for clarity.

Figure 4

Proposed mechanism of CA esterase function, which converts Aspirin to SA. (A) Aspirin binds through the zinc bound hydroxyl orienting its acetyl group to how acetate binds in CAII (PDB: 1CAY). (B) The ester bond in Aspirin is cleaved via nucleophilic attack. (C) Once the ester bond is cleaved, acetate is generated and briefly is bound to the zinc. (D) A solvent molecule replaces acetate on the zinc and acetate is released to bulk solvent. (E) Salicylic acid rotates to bind its carboxylic acid moiety to the zinc bound solvent, stabilizing it within the active site.