Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5521
using PRISM 3, as reported earlier,20,22,23 and represent the
mean from at least three different determinations.
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Crystallization, X-ray Data Collection, and Refinement. Crys-
tals of the complex between hCA II and spermine 4 were
obtained by using the hanging-drop method for cocrystallizing
the protein with the ligand, as previously described.20 Drops
containing 5 μL of 20 mg/mL hCA II in 50 mM Tris-HCl,
buffer, pH 7.4, were mixed with 5 μL of precipitant buffer (2.4 M
(NH4)2SO4 in 50 mM Tris-HCl, pH 7.4, and 1 mM sodium
4-(hydroxymercury) benzoate) with added 5 mM spermine 4
and equilibrated over a reservoir of 1 mL of precipitant buffer. A
monochromatic experiment at the Cu R wavelength was per-
formed on a crystal of hCA II grown in the presence of 4 by the
rotation method on a PX-Ultra sealed-tube diffractometer
(Oxford Diffraction) at 100 K. The crystal belonged to space
group P21 (a = 41.4; b = 42.1; c = 72.2; β = 104.3). Data were
processed with CrysAlis RED (Oxford Diffraction 2006).28 The
structure was analyzed by difference Fourier technique, using
the PDB file 1CA225b as starting model. The refinement was
carried out with the program REFMAC529 model building, and
map inspections were performing using the COOT program.30
The correctness of the stereochemistry was checked using the
program PROCHECK.31 The refinement statistics of the final
model of the hCA II-spermine complex are shown in Table 2.
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Part 3. Structure-activity correlations for a series of isozyme II
activators. J. Pharm. Sci. 1994, 83, 768–779. (b) Temperini, C.; Vullo,
D.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase activators.
Activation of isoforms I, II, IV, VA, VII, and XIV with L- and
D-phenylalanine and crystallographic analysis of their adducts with
isozyme II: sterospecific recognition within the active site of an enzyme
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high affinity isozymes I, II, and IV activators, incorporating a
β-alanyl-histidine scaffold. Carbonic anhydrase activators: human
isozyme II is strongly activated by oligopeptides incorporating
the carboxyterminal sequence of the bicarbonate anion exchanger
AE1. J. Med. Chem. 2002, 45, 284–291. (b) Ilies, M.; Banciu, M. D.;
Ilies, M. A.; Scozzafava, A.; Caproiu, M. T.; Supuran, C. T. Carbonic
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Acknowledgment. This research was financed in part by
a grant of the 6th framework programme of the European
Union (DeZnIT project) and one from the 7th framework pro-
gramme (Metoxia). K.K.’s work is supported by the Academy
ꢁ
of Finland and the Sigrid Juselius Foundation. Thanks are
addressed to Dr. Giuseppina De Simone for helpful discussion
with the X-ray crystallographic part of our work.
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