sodium-nitrite and sulfamic-acid

Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The β-class CAs (β-CAs) are expressed in prokaryotes, fungi, plants, and more recently have been isolated in some animals. The β-CA class is divided into two subclasses, termed type I and II, defined by pH catalytic activity profile and active site structural configuration. Type I β-CAs display catalytic activity over a broad pH range (6.5-9.0) with the active site zinc tetrahedrally coordinated by three amino acids and a hydroxide/water. In contrast, type II β-CAs are catalytically active only at a pH 8 and higher where they adopt a functional active site configuration like that of type I. However, below pH 8 they are conformationally self-inactivated by the addition of a fourth amino acid coordinating the zinc and thereby displacing the zinc bound solvent. We have determined the structure of psCA3, a type II β-CA, isolated from Pseudomonas aeruginosa (P. aeruginosa) PAO1 at pH 8.3, in its open active state to a resolution of 1.9 Å. The active site zinc is coordinated by Cys42, His98, Cys101 and a water/hydroxide molecule. P. aeruginosa is a multi-drug resistant bacterium and displays intrinsic resistance to most of the currently used antibiotics; therefore, there is a need for new antibacterial targets. Kinetic data confirm that psCA3 belongs to the type II subclass and that sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. In vivo studies identified that among six tested inhibitors representing sulfonamides, inorganic anions, and small molecules, acetazolamide has the most significant dose-dependent inhibitory effect on P. aeruginosa growth.

Topics: Bacterial Proteins; Binding Sites; Boronic Acids; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Catalytic Domain; Crystallography, X-Ray; Dimerization; Drug Resistance, Multiple, Bacterial; Hydrogen-Ion Concentration; Kinetics; Molecular Dynamics Simulation; Protein Structure, Tertiary; Pseudomonas aeruginosa; Recombinant Proteins; Sulfonamides; Sulfonic Acids

Carbonic anhydrase (CA; EC 4.2.1.1) was purified from the gill of the teleost fish Dicentrarchus labrax (European seabass). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 84.9-fold with a yield of 58%, and a specific activity of 838.9 U/mg proteins. It has an optimum pH at 8.0; an optimum temperature at 10°C. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate. The following anions, H₂NSO₃⁻, I⁻, SCN⁻, NO₃⁻, NO₂⁻, N₃⁻, Br⁻, Cl⁻, SO₄²⁻, and F⁻ showed inhibitory effects on the enzyme. Sulfamic acid, iodide, and thiocyanate exhibited the strongest inhibitory action, in the micromolar range (K(i)s of 87-187 μM). NO₃⁻, NO₂⁻ and N₃⁻ were moderate inhibitors, whereas other anions showed only weak actions. All tested anions inhibited the enzyme in a competitive manner. Our findings indicate that these anions inhibit the fish enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various anions differs significantly between the fish and mammalian CAs.

Topics: Animals; Anions; Bass; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Kinetics; Sulfonic Acids; Thiocyanates