Human carbonic anhydrase II-cyanate inhibitor complex: putting the debate to rest

Abstract

The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO2. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5 Å resolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO2-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (∼40 µM) were measured using (18)O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO2 substrate-binding site.

Keywords: carbonic anhydrase; cyanate; α-class CA.

Figure 1

Coordination states of the zinc ion in the active site of CA II. (a) Tetrahedral coordination with azide. (b) Pentacoordination with thiocyanate. (c) CO₂ binding in the active site prior to nucleophilic attack by ZnOH⁻ (tetrahedral coordination maintained).

Figure 2

Stick diagrams of (a) wild-type and (b) V207I CA II in complex with cyanate. F _o − F _c electron-density map contoured at 3σ. The hydrogen-bonding distance (2.9 Å) between the main-chain amide of Thr199 and the O atom of cyanate is indicated as a dashed line. (c) Overlay of wild-type (dark grey) and V207I (light grey) CA II in complex with cyanate. Active-site residues are as labelled and the zinc is represented as a gray sphere. This figure was produced using PyMOL (v.1.5.0.4; Schrödinger).

Figure 3

Overlay of wild-type CA II in complex with cyanate (dark gray) and CA II in complex with CO₂ (white; Domsic et al., 2008; PDB entry 3d92). The root-mean-squared deviation between all C^α atoms is 0.25 Å. This figure was produced using PyMOL (v.1.5.0.4; Schrödinger).