Comparison of solution and crystal properties of Co(II)-substituted human carbonic anhydrase II.
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 pK(a) 8.4 for the ionization of the metal-bound water in the crystal. This is higher by about 1.2 pK(a) units than the pK(a) near 7.2 for Co(II)-CA II in solution. This effect is attributed to a nonspecific ionic strength effect of 1.4M citrate in the precipitant solution used in the crystal growth. A pK(a) of 8.3 for the aqueous ligand of the cobalt was measured for Co(II)-HCA II in solution containing 0.8M 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 1.5-1.6A 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.
Avvaru
Balendu Sankara
BS
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA.
Arenas
Daniel J
DJ
Tu
Chingkuang
C
Tanner
D B
DB
McKenna
Robert
R
Silverman
David N
DN
D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry; D018360:Crystallography, X-Ray; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007700:Kinetics; D008958:Models, Molecular; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013053:Spectrophotometry; D055672:Static Electricity; D015032:Zinc / Q000737:chemistry
D011994:Recombinant Proteins; D012996:Solutions; D003035:Cobalt; D024402:Carbonic Anhydrase II; D015032:Zinc