Article ID Journal Published Year Pages File Type
1925958 Archives of Biochemistry and Biophysics 2010 7 Pages PDF
Abstract

The visible absorption of crystals of Co(II)–substituted human carbonic anhydrase II (Co(II)–HCA II) were measured over a pH range of 6.0–11.0 giving an estimate of pKa 8.4 for the ionization of the metal-bound water in the crystal. This is higher by about 1.2 pKa units than the pKa near 7.2 for Co(II)–CA II in solution. This effect is attributed to a nonspecific ionic strength effect of 1.4 M citrate in the precipitant solution used in the crystal growth. A pKa of 8.3 for the aqueous ligand of the cobalt was measured for Co(II)–HCA II in solution containing 0.8 M citrate. Citrate is not an inhibitor of the catalytic activity of Co(II)–HCA II and was not observed in crystal structures. The X-ray structures at 1.5–1.6 Å resolution of Co(II)–HCA II were determined for crystals prepared at pH 6.0, 8.5 and 11.0 and revealed no conformational changes of amino-acid side chains as a result of the use of citrate. However, the studies of Co(II)–HCA II did reveal a change in metal coordination from tetrahedral at pH 11 to a coordination consistent with a mixed population of both tetrahedral and penta-coordinate at pH 8.5 to an octahedral geometry characteristic of the oxidized enzyme Co(III)–HCA II at pH 6.0.

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