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.

Reconsidering anion inhibitors in the general context of drug design studies of modulators of activity of the classical enzyme carbonic anhydrase

“… 27–30 . Such interesting and valuable studies were mainly reported by the groups of Coleman 29 , 30 and Bertini-Scozzafava 27 , 28 , which reinforced the earlier conclusions 3–5 that the anion CAIs coordinate to the metal ion in the enzyme active site in a tetrahedral or more rarely, a trigonal bipyramidal geometry 26–30 , as shown schematically in Figure 2 for some anion inhibitors, again using hCA II as model enzyme and high-resolution X-ray crystallography 31–33 .…”

“… Representation of the superimposed hCA II adducted to cyanate (cyan, pdb 4E5Q 31 ) bicarbonate (pink, pdb 2VVB 24 ) urea (as anion) (white, pdb 1BV3 [39]), and trithiocarbonate (light blue, pdb 3K7K 40 ) The zinc ion (grey sphere), its protein ligands (His94, 96 and 119) and gate-keeping residues (Thr199, Glu106) are also shown. …”

“… Representation of the superimposed X-ray crystallographic data for hCA II adducts with cyanate (cyan, pdb 4E5Q 31 ) bromide (magenta, pdb 1RAZ 32 ) azide (blue, pdb 1RAY 32 ) O 2 (presumably as superoxide anion or radical anion; red, pdb 5EOI 33 ) with a tetrahedrally coordinated active site zinc ion (grey sphere). The key active site zinc ligands (His94, 96 and 119) and “gate-keeping” residues (Thr199, Glu106) are also shown.…”

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

“…Cyanate was reported to bind in a CO 2 binding site, where Zn-bound water kept the position as a metal ligand (Lindahl, et al ., 1993). Different ways of cyanate binding were found in the structures of WT CA II and V207I mutant with cyanate (West et al ., 2014). The anion was bound directly to Zn finishing a tetrahedral coordination sphere.…”

Investigation of pesticides on honey bee carbonic anhydrase inhibition

“…The best example is the report by Liljas' group that cyanide and cyanate do not coordinate the metal ion from CA active site 44 . Subsequent work from other laboratories showed those data to be false, as both cyanate and cyanide were observed coordinated to the metal ion, as most other anion inhibitors investigated to date 45,46 . Furthermore, cyanate was also shown to be a suicide substrate that can be hydrolysed by the CA activity with the formation of carbamate 45 .…”