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PDBsum entry 1bv3
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Carbonic anhydrase catalyzes cyanamide hydration to urea: is it mimicking the physiological reaction?
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Authors
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F.Briganti,
S.Mangani,
A.Scozzafava,
G.Vernaglione,
C.T.Supuran.
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Ref.
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J Biol Inorg Chem, 1999,
4,
528-536.
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PubMed id
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Abstract
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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, CO(2), 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 x
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.
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