Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction? (original) (raw)

Abstract

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±9 mM 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(II)-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=2.5×10–5 s–1 M–1). A mechanism to 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.

Access this article

Log in via an institution

Subscribe and save

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

Author information

Authors and Affiliations

  1. Università di Firenze, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121 Florence, Italy e-mail: scoz@as1.cerm.unifi.it Tel.: +39–055–2757551, Fax: +39–055–2757555, , , , , , IT
    Fabrizio Briganti, A. Scozzafava, Guido Vernaglione & Claudiu T. Supuran
  2. Università di Siena, Dipartimento di Chimica, Pian dei Mantellini 44, I-53100 Siena, Italy, , , , , , IT
    Stefano Mangani

Authors

  1. Fabrizio Briganti
    You can also search for this author inPubMed Google Scholar
  2. Stefano Mangani
    You can also search for this author inPubMed Google Scholar
  3. A. Scozzafava
    You can also search for this author inPubMed Google Scholar
  4. Guido Vernaglione
    You can also search for this author inPubMed Google Scholar
  5. Claudiu T. Supuran
    You can also search for this author inPubMed Google Scholar

Additional information

Received: 26 February 1999 / Accepted: 25 May 1999

Rights and permissions

About this article

Cite this article

Briganti, F., Mangani, S., Scozzafava, A. et al. Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction?.JBIC 4, 528–536 (1999). https://doi.org/10.1007/s007750050375

Download citation