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The three- Two major forms of carbonic anhydrase occur in human erythrocytes (10): the low activity form carbonic anhydrase B, and the high activity form carbonic anhydrase C. Carbonic anhydrase B is found in greater quantity than carbonic anhydrase C. The complete amino-acid sequences of the two isozymes have been established (11)(12)(13)(14) and show a great deal of homology. The three-dimensional structure of carbonic anhydrase C has been established to 2-A resolution (15, 16); the present article presents the high-resolution structure determination of carbonic anhydrase B. MATERIALS AND METHODSCrystallization and Heavy Atom Derivatives. Human carbonic anhydrase B was isolated from erythrocytes (kindly provided by the University Hospital, Uppsala) by mild methods (17). The enzyme was further purified by electrophoresis and on a Sulfur-ethylsephadex C-50 ion exchange column. Crystals of the native enzyme were prepared from 2.3 M ammonium sulfate, pH 8.7, by a seeding technique in small, thick-walled glass capillaries as described (18). Heavy atom derivatives were prepared by soaking native enzyme crystals for at least a week in 2.3 M ammonium sulfate solution, pH 8.7, containing 1 mM heavy atom salts, K2Au(CN)2, * This is paper II in a series. In all 30 layers, zero and upper levels were collected to cover about 85% of the reflections within the 2-A sphere. Intensity measurements were performed by a fully automatic microdensitometer constructed by Ing. V. Klimecki of the Department of Chemistry, University of Uppsala, in collaboration with our group. The program system used for data processing was developed by one. of us (K.K.K.) and is similar to that described by Jarup et al. (21).All the symmetry-related reflections were averaged, and the average amplitudes were used in subsequent calculations. The reflections in the 10-A sphere and reflections below a threshold were omitted.. Out of the 14,000 unique reflections thus measured, about 10,500 reflections were used in the refinements of heavy atom parameters, phase angle calculations, and electron density maps.Heavy Atom Parameters and Phase Angle Calculation. The heavy atom positional parameters were initially determined from difference Patterson and difference Fourier calculations in two orthogonal projections. A single site mercury derivative (18) was used in the two centrosymmetric projections to calculate the signs used in the difference Fouriers from which the principal heavy atom positions were determined for the other derivatives. Subsequent cycles of least-squares refinement and difference Fouriers included all the derivatives. This procedure did not introduce any serious biasing effect due to the heavy atom derivatives concerned. Threedimensional difference Patterson and correlation functions (22) were calculated to verify the correct assignment of the heavy atom positions. Alternate cycles of phase angle calculation and least-squares refinement were performed for relative scale factor, overall isotropic temperature factor, positional paramet...

The structure of human erythrocyte carbonic anhydrase I has been refined to a final R value of 19% to 2-A resolution by a combination of least squares refinement and model fitting in a three-dimensional graphics display. About 300 solvent atoms have been located bound to the protein molecule. An interesting hydrogen bond network involving Zn2+, the liganded solvent, side chain groups of Thr-199, Glu-106, Thr-7, and His-64 through two solvent molecules have been found that may be important for the catalytic mechanism of the carbonic anhydrase.

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