The interaction of human carbonic anhydrase (hCA) isozymes I and II with cyanamide, a linear molecule isoelectronic with the main physiological substrate of the enzyme, CO2, was investigated through spectroscopic, kinetic, and X-ray crystallographic studies. We show here that cyanamide is hydrated to urea in the presence of CAs, and that it also acts as a weak non-competitive inhibitor (K-I=61 +/- 3 mM and 238+/-9mM for hCA II and hCA I, respectively) towards the esterasic activity of these enzymes, as tested with 4-nitrophenyl acetate. Changes in the spectrum of the Co(III)-hCA II derivative observed in the presence of cyanamide suggest that it likely binds the metal ion within the CA active site, adding to the coordination sphere, not substituting the metal-bound solvent molecule. It thereafter undergoes a nucleophilic attack from the metal-bound hydroxide ion, forming urea which remains bound to the metal, as observed in the X-ray crystal structure of hCA II soaked in cyanamide solutions for several hours. The urea molecule is directly coordinated to the active site Zn(II) ion through a protonated nitrogen atom. Several hydrogen bonds involving active site residues Thr199 and Thr200 as well as three water molecules (Wat99, Wat122, and Wat123) further stabilize the urea-hCA II adduct. Kinetic studies in solution further proved that urea acts as a tight binding inhibitor of the two isozymes hCA I and hCA II, with very slow binding kinetics (k(on) =2.5 x 10(-5) s(-1)M(-1)). A mechanism eo explain the hydration process of cyanamide by CAs, as well as the tight binding of urea in the active site, is also proposed based on the hypothesis that urea is deprotonated when bound to the enzyme. Cyanamide is thus the first true suicide substrate of this enzyme for which binding has been documented by means of X-ray crystallographic and spectroscopic studies.
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|Titolo:||Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction?|
|Citazione:||F., B., Mangani, S., A., S., G., V., & C. T., S. (1999). Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction?. JBIC, 4(5), 528-536.|
|Appare nelle tipologie:||1.1 Articolo in rivista|
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