Mechanism of cyanamide hydration catalyzed by carbonic anhydrase II suggested by cryogenic X-ray diffraction

被引:44
作者
Guerri, A
Briganti, F
Scozzafava, A
Supuran, CT
Mangani, S
机构
[1] Univ Siena, Dept Chem, I-53100 Siena, Italy
[2] Univ Florence, Lab Chim Inorgan & Bioinorgan, I-50121 Florence, Italy
关键词
D O I
10.1021/bi000937c
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The three-dimensional structure of a possible intermediate in the hydration reaction of cyanamide to urea catalyzed by human carbonic anhydrase II (hCAII) has been determined by cryocrystallographic techniques. The crystal structure shows that two different adducts are formed under the experimental conditions and that they have different occupancy in the crystal. The high occupancy form consists of a binary hCAII-cyanamide complex where the substrate has replaced the zinc-bound hydroxide anion present in the native enzyme, maintaining the tetrahedral geometry around the metal ion. The second, low-occupancy form consists of a hCAII-cyanamide-water ternary complex where the catalytic zinc ion, still being bound to cyanamide, is approached by a water molecule in a five-coordinate adduct. While the first form can be considered a nonproductive complex, the second form may represent an intermediate state of the catalyzed reaction where the water molecule is about to perform a nucleophilic attack on the zinc-activated cyanamide substrate. The structural evidence is consistent with the kinetic data previously reported about this recently described hydrolytic reaction catalyzed by hCAII, and indicates that a different mechanism with respect to that generally accepted for the physiologic carbon dioxide hydration reaction may be adopted by the enzyme, depending on the substrate chemical properties.
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页码:12391 / 12397
页数:7
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