Crystal Structure of the "cab"-type beta  Class Carbonic Anhydrase from the Archaeon Methanobacterium thermoautotrophicum*

Pavel StropDagger , Kerry S. Smith§, Tina M. Iverson, James G. Ferry§||, and Douglas C. Rees**

From Dagger  Biochemistry Option, California Institute of Technology, Pasadena, California 91125, the § Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, and the  Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, and the || Center for Microbial Structural Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication, October 9, 2000, and in revised form, November 26, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The structure of the "cab"-type beta  class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) has been determined to 2.1-Å resolution using the multiwavelength anomalous diffraction phasing technique. Cab exists as a dimer with a subunit fold similar to that observed in "plant"-type beta  class carbonic anhydrases. The active site zinc is coordinated by protein ligands Cys32, His87, and Cys90, with the tetrahedral coordination completed by a water molecule. The major difference between plant- and cab-type beta  class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes buffer molecule bound 8 Å away from the active site zinc, which suggests a possible proton transfer pathway from the active site to the solvent.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Carbonic anhydrases (CAs)1 are Zn2+-containing enzymes that catalyze the reversible hydration of CO2 (1). With turnover numbers approaching 106 s-1, CAs are among the fastest known enzymes. CAs have been found in most types of organisms, including mammals, plants, algae, bacteria, and archaea (2). Based on the amino acid sequences, CAs can be assigned to one of three independently evolved classes, designated alpha , beta , and gamma  (3).

The alpha  class contains all mammalian CAs, as well as some CAs from algae and bacteria (3, 4). alpha -CAs play important roles in respiration, secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pH homeostasis, and ion exchange (5, 6). Crystal structures of alpha -CA have revealed a monomer organized around a 10-stranded, predominantly antiparallel beta -sheet (7-13). The catalytically active zinc is coordinated by three histidines and one water molecule.

gamma -CA has thus far been isolated and characterized only from the methanoarchaeon Methanosarcina thermophila (14-16), where it is proposed to facilitate the transport of CH3COO- and to convert CO2 to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> outside the cell to assist the removal of excess CO2 generated during the growth of this organism on acetate. The M. thermophila gamma -CA exists as a trimer, with the active site located at the interface between two subunits. Each subunit is organized around a left-handed beta -helix that is completely distinct from the alpha -CA fold, although the active site is also coordinated by three histidines, along with two water molecules (17, 18).

The beta  class includes CAs from plants, algae, bacteria, and archaea (2, 3). In higher plants, beta -CAs play an important role in photosynthesis, by concentrating CO2 in the proximity of ribulose bisphosphate carboxylase/oxygenase for CO2 fixation (19). The purification and characterization of carbonic anhydrase (Cab) from the thermophilic Methanobacterium thermoautotrophicum extends this class into the archaea (20). Cab is at the phylogenetic extreme of the beta  class carbonic anhydrases and forms an exclusively prokaryotic clade consisting primarily of sequences from Gram-positive bacteria (2). In the obligate chemolithoautotroph M. thermoautotrophicum, Cab converts CO2 to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, suggesting that the physiological role of this enzyme may be to provide HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to enzymes important in CO2 fixation pathways of the microbe (21, 22).

beta class carbonic anhydrases can be further divided into "plant"- and "cab"-type, based on the active site sequence conservation (20, 23) (Fig. 1). Two crystal structures of plant-type beta -CA were recently reported from Porphyridium purpureum (P. purpureum beta -CA) (24) and Pisum sativum (P. sativum beta -CA) (23). The basic fold of beta -CA consists of a four-stranded, parallel beta -sheet core with alpha -helices forming right-handed cross-over connections (23, 24). The oligomerization state of beta -CA is variable, however, and P. purpureum beta -CA and P. sativum beta -CA exist as a dimer and octamer, respectively, although the dimer of P. purpureum beta -CA resembles a tetramer, where two monomers are fused together. In contrast to the protein ligation by three histidines observed in alpha - and gamma -CAs, the active site zinc in beta -CAs is coordinated by two cysteines and one histidine, as anticipated from extended X-ray absorption fine structure spectroscopy studies (21, 25, 26). The fourth ligand is different in the two beta -CAs structures. In the P. sativum beta -CA structure, an acetate molecule is bound to the zinc, whereas in the P. purpureum beta -CA structure, the side chain of aspartic acid (Asp151) acts as the fourth ligand. In the P. sativum beta -CA structure, this conserved Asp interacts with a conserved Arg (Fig. 1).


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Fig. 1.   Alignment of beta -CA sequences. Cab, M. thermoautotrophicum; PPnterm, P. purpureum N-terminal domain; PPcterm, P. purpureum C-terminal domain; P.S., P. sativum; ECcynT, E. coli. Zinc ligands are colored yellow, the conserved Asp/Arg pair is red, and residues differentiating cab-type and plant-type are blue.

Apart from the conserved zinc ligands and the Asp/Arg pair, the active site of Cab differs significantly from the plant-type beta -CAs. Cab active site residues His23, Met33, Lys53, Ala58, and Val72 are replaced in the plant-type beta -CA by Gln, Ala, Phe, Val, and Tyr, respectively. Residues that are equivalent to Cab residues Met33, Lys53, Ala58, and Val72 make up the hydrophobic pocket in the P. sativum and P. purpureum beta -CA structures. Substitutions of the two aromatic side chains by Lys and Val in Cab suggest a significant redesign of the hydrophobic pocket in cab-type beta -carbonic anhydrase. Cab has a CO2 hydration activity with a kcat of 1.7 × 104 s-1 and Km for CO2 of 2.9 mM at pH 8.5 and 25 °C (20). Cab is inhibited by iodide, nitrate, and azide; however, in contrast to plant-type beta -CAs, chloride and sulfate have no effect on Cab activity. These active site substitutions, together with the different effects of inhibitors, imply that there might be mechanistically relevant differences in the organization of the active sites between cab-type and plant-type enzymes. Here we present the first structure of the cab-type beta -carbonic anhydrase from thermophilic methanoarchaeon M. thermoautotrophicum, determined at 2.1-Å resolution.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Crystallization-- Cab was overexpressed in Escherichia coli and purified by a heat denaturation step followed by ion exchange chromatography as previously described (20). Crystals were grown by the hanging drop method at 22 °C using a 5 mg/ml protein solution and a precipitant solution containing 100 mM Hepes, pH 7.5, 35% ethanol, 12% 2-methyl-2,4-pentanediol, and 50 mM calcium acetate. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 54.9 Å, b = 113.2 Å, c = 156.2 Å and three dimers per asymmetric unit. Crystals were transferred stepwise to mother liquor solution containing 30% 2-methyl-2,4-pentanediol as a cryoprotectant and flash frozen.

Data Collection and Processing-- A three-wavelength multiwavelength anomalous dispersion data set was collected at -160 °C on beamline 9-2 of the Stanford Synchrotron Research Laboratory with an ADSC charge-coupled device detector. The fluorescence spectrum measured around the zinc edge of a single crystal was used to select the inflection point (lambda  = 1.2832 Å), the absorption edge (lambda  = 1.282 Å), and a high energy remote wavelength (lambda  = 1.033 Å) for optimization of the anomalous signal. All data were reduced using DENZO and scaled using SCALEPACK (27) (Table I).

                              
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Table I
Data collection statistics
Numbers in parentheses indicate values for highest resolution bin (2.38-2.30 Å for lambda 1, 2.49-2.4 Å for lambda 2, and 2.17-2.10 Å for lambda 3. cen, centric; acen, acentric; anom, anomalous.

Phase Determination-- The structure was determined by multiwavelength anomalous dispersion using the signal from only the intrinsic zinc atoms. The program SOLVE (28) was used to find the positions of the heavy atoms using the three wavelength multiwavelength anomalous dispersion data set using data from 20- to 2.4-Å resolution. Two zinc ions were identified and when used for phasing yielded a figure of merit of 0.44. Four additional zinc sites were located in an anomalous difference Fourier map, yielding a figure of merit of 0.54. Each of the six zinc sites corresponded to a Cab monomer, which are organized into three tight dimers in the asymmetric unit. The initial noncrystallographic symmetry (NCS) transformations were established by the relationships between the dimers, and the initial mask was calculated with the program NCSMSK (29). The program DM (30) was used for NCS averaging of the electron density maps, solvent flattening, and phase extension from 2.4- to 2.1-Å resolution. The resulting map was of good quality and allowed building of most of the protein.

Refinement-- Alternate cycles of manual model building using the program O (31) and positional and individual B-factor refinement with the program CNS (32) reduced the R and Rfree to 21.1 and 24.6% respectively, where Rfree is calculated for 5% (2875) of the reflections in the resolution range 18-2.1 Å. The model was initially refined with strict NCS restraints, which were released later in the refinement. The r.m.s. deviation of bond lengths and angles are 0.013 Å and 1.7° respectively, with 87.9% in the most allowed region and 11.1% in the additionally allowed region of the Ramachandran plot. The average B-factors are 28.6 Å2 (main chain), 32.4 Å2 (side chains), 18.6 Å2 (zinc atoms), and 36.3 Å2 (solvent). An average B-factor of 30.5 Å2 is calculated for all protein atoms. The final model contains 7543 protein atoms, 3 Hepes molecules, 6 zinc atoms, 6 calcium atoms, and 409 water molecules, for a total of 8015 atoms (Table II).

                              
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Table II
Refinement statistics


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Structural Organization of Cab-- The overall fold of the Cab monomer (Fig. 2A) consists of a four-stranded parallel beta -sheet core with strand order 2-1-3-4. Monomers in each dimer are related by a 2-fold axis centered between strands beta 2. The overall dimensions of the dimer are ~40 × 45 × 50 Å (Fig. 2B). Three dimers, designated AB, CD, and EF, are present in the asymmetric unit. In the structurally conserved beta -sheet region (residues 26-32, 52-57, 80-88, 149-157, and 163-167), the r.m.s. deviations in Calpha positions between the dimers average 0.24 Å. Using Calpha positions for residues 24-170, the corresponding r.m.s. deviations are 0.34 Å between dimers AB and CD, 1.0 Å between dimers AB and EF, and 1.1 Å between dimers CD and EF. The relatively large r.m.s. deviations for the latter two pairs of dimers reflect the poor ordering for residues 91-126 in monomer E. Excluding these residues, the r.m.s. deviations drop to ~0.28 Å. The r.m.s. deviation in the beta -sheet region between the A and B, C and D, and E and F subunits of the Cab are ~0.6 Å, while the r.m.s. deviation for all Calpha between the subunits are ~0.8 Å.


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Fig. 2.   Structure of Cab. A, ribbon diagram of the A monomer of Cab. The color changes from blue (N terminus) to red (C terminus). B, dimer of Cab. Monomer A is shown in magenta, monomer B is shown in blue, and zinc atoms are shown in gray. Monomer A is shown in the same orientation for both A and B. Hepes bound to subunit A is shown in a ball-and-stick representation. The images were created with BOBSCRIPT (45) and RASTER3D (46).

The regions with the largest conformational variation include the N-terminal residues 1-23 (involved in crystal packing), residues 92-95, and residues 120-125. While the N-terminal residues 1-23 are well defined in monomers A, C, and E, residues 13-23 are disordered in monomers B, D, and F. Residues 92-95 and 120-125 form a hinge region for helices alpha 4 and alpha 5 and are well ordered in monomers B and D, slightly disordered in monomers A, C, and F, and disordered in monomer E. The variability in region 90-125 between the six crystallographically independent monomers suggests that these residues are conformationally mobile. Overall, with the exception of the noted regions, the three dimers in the asymmetric unit are very similar. Unless stated otherwise, only dimer AB will be used in future discussions.

Six calcium ions were located on the surface of Cab and at the crystal packing interfaces between Cab dimers. Most likely, these calcium ions do not play any structural or catalytical role and are a result of the crystallization conditions that contained 50 mM calcium acetate.

Cab Oligomerization-- beta -CAs have been found in different oligomeric states ranging from dimers (Oryza sativa, P. purpureum) to tetramers (E. coli) to octamers (P. sativum) (20, 23, 24, 33). Although analytical ultracentrifugation results suggest that Cab exists as a homotetramer (21), Cab appears to form a dimer in the crystal. There are numerous interactions stabilizing the dimer involving residues in beta -strand beta 2 and helices alpha 2, alpha 3, alpha 4, and alpha 5. Hydrogen bonds between residues 56 and 57 from strand beta 2 in both subunits result in a formation of a 10-stranded beta  sheet. Helices alpha 4 and alpha 5 extend out and make extensive contacts with the other monomer in the dimer (Fig. 2B). The interface area between these two subunits was found to be ~2110 Å2/subunit, which is ~21% of the total (~10,000 Å2) monomer accessible surface area as calculated with GRASP (34). Residues A2-A23 pack against a symmetry-related molecule burying 860 Å2, resulting in the formation of a continuous ribbon through the crystal (Fig. 3). Residues B2-B12 most likely fold back and pack against the B monomer in the same way that A2-A12 packs against the symmetry-related molecule. Residues B13-B23 have no visible electron density and are disordered. A similar type of crystal packing forming a continuous ribbon has been observed in the sterile alpha  motif domain of the human EphB2 receptor and, together with other evidence, was proposed to be functionally important (35). The crystal packing interaction seen in Cab can also be described as a linear (open-ended) domain swapped oligomer, where the swapped domain consists of a 12-residue alpha -helix (36, 37). It is unclear whether residues 1-24 could facilitate the formation of a higher oligomerization state under physiological conditions or whether residues A2-A23 also fold back and pack against monomer A. Based on modeling considerations, although the N-terminal region is highly flexible, formation of a Cab tetramer similar to the one seen in the P. purpureum structure is unlikely in the absence of conformational changes due to steric clashes of helices alpha 4 and alpha 5. Other contacts between Cab dimers in the crystal are relatively small (<= 540 Å2) and, based on their complementarity and size (38, 39), are also unlikely to support formation of stable tetramers.


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Fig. 3.   Stereo view of the crystal packing of Cab showing the continuous ribbon created by packing of helix alpha 1 against a crystallographically related molecule. Monomer A is in magenta, with helix alpha 1 in green. Monomer B is in blue, with helix alpha 1 in red.

Comparison of Cab and P. sativum and P. purpureum Structures-- The fold of Cab is similar to that of the P. sativum and P. purpureum beta -CAs (Fig. 4). While the beta -sheet core is conserved, significant secondary structure differences are evident, mostly in the regions at the N terminus, at the C terminus, and in the region containing residues 90-125. The N termini of the P. sativum and P. purpureum beta -CA structures extend out, forming a long helix that packs against a second monomer, making additional dimer interactions. In Cab, the N terminus is involved in crystal packing and does not adopt the same conformation observed in the P. sativum and P. purpureum beta -CA structures. Cab is one of the smallest beta -CAs known and lacks an extended C terminus. In the P. sativum beta -CA structure, the C terminus forms a long beta -strand that mediates octamerization. In Cab, residues 90-125 form two helices (alpha 4, alpha 5) that project out to cover the second monomer (Fig. 2B) and fold back to start helix alpha 6. In both the P. sativum and P. purpureum beta -CA structures, this segment is longer, forms three helices instead of two, and folds back earlier to create two additional turns of helix alpha 6. In the central beta -sheet region, the r.m.s. deviations in Calpha positions between Cab and P. sativum and P. purpureum structures are 0.62 and 0.56 Å, respectively.


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Fig. 4.   Stereo diagram showing the superposition of a backbone trace of Cab (yellow), P. sativum beta -CA (green), and P. purpureum beta -CA (blue). The main differences are at the N terminus, C terminus, and in the alpha 4, alpha 5 region.

Active Site-- The active site cleft is located at the C terminus of the parallel beta -sheet and is largely sequestered from solvent. Each Cab subunit contains one zinc atom that resides at the interface of the two monomers (Fig. 2B), although the coordination residues (Cys32, Cys90, and His87) originate from the same monomer. One water molecule completes the tetrahedral coordination sphere of the zinc. Although the crystallization conditions contained 50 mM calcium acetate, no acetate was found in the active site, unlike in the P. sativum beta -CA structure (Fig. 5A). The average coordination distances from the six active sites are as follows: Cys32 Sgamma -Zn (2.42 ± 0.03 Å), His87 Nepsilon -Zn (2.11 ± 0.04 Å), Cys90 Sgamma -Zn (2.40 ± 0.04 Å), and H2O-Zn (2.15 ± 0.06 Å). The four zinc ligands form a number of hydrogen bonds with the surrounding residues. His87 hydrogen-bonds with the carbonyl oxygen of residue Thr88; Cys32 Sgamma hydrogen-bonds with the amide nitrogens of residues Lys22, Asp34, and Gly59; Cys90 hydrogen-bonds with the amide nitrogen of residue Met92; and the coordinating water molecule makes a hydrogen bond to Asp34. Arg36 forms the conserved Asp/Arg pair with Asp34 and also interacts with Asp89 and a water molecule.


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Fig. 5.   Stereo view of the zinc environment in Cab. A, the simulated annealing (|Fo- |Fc|) omit map, calculated after omitting the four ligands and the zinc, is contoured at 4sigma (blue) and 15sigma (red). A simulated annealing omit map calculated with only the coordinating water molecule omitted is shown contoured at 4sigma (green). B, Hepes binding to the active site of subunit A. Hepes is in purple, with hydrogen bonds shown as dotted lines. The loop shown in green would sterically clash with Hepes if Hepes bound to the active site of subunit B as it is observed to bind to the active site of subunit A.

Rather unexpectedly, the two active sites A and B in the Cab dimer exhibit significant differences, which are reflected in the r.m.s. deviations between monomers in a dimer (~0.65 Å) being consistently larger than between the equivalent monomers in different dimers such as A and C, or B and D (0.24 Å). The active sites in monomers A, C, and E have a Hepes buffer molecule bound near the active site. The sulfate group of the Hepes is located ~8 Å from the zinc atom, and the sulfate oxygens hydrogen bond with Lys53 Nzeta , Ser35 Ogamma , and the amide nitrogen of Ser35 (Fig. 5B). The equivalent to Ser35 is present in both plant-type beta -CAs and in Cab, while Lys53 is unique to the cab-type beta -CAs. Asp34, which makes a hydrogen bond to the zinc-coordinating water molecule, is also within hydrogen bonding distance (~3.0 Å) to the Hepes sulfate group. Another difference between the two active sites is the conformation of residues 13-24. In the active site of subunit A, residues B13-B24 are disordered and have no visible electron density. In the active site of subunit B, however, residues A13-A24 are well defined and extend out to participate in crystal packing. Superposition of residues in the active sites of subunits A and B (Fig. 5B) indicates that the Hepes molecule would sterically clash with residues Arg16 and Asp17 if residues 13-24 adopted the same conformation as in active site B.

Comparison of P. sativum beta -CA and Cab Active Sites-- The zinc-coordinating residues (Cys32, His87, and Cys90) of Cab and P. sativum beta -CA superimpose closely (Fig. 6A). The water molecule serving as the fourth zinc ligand in Cab adopts a position similar to the O1 of the acetate ligand in the P. sativum beta -CA structure. The conserved residue Asp34 is held in place by forming two hydrogen bonds to Arg36, as do the corresponding residues Asp162 and Arg164 in the P. sativum beta -CA. The following five active site substitutions distinguish the cab-type and plant-type beta -carbonic anhydrases: H23Q, M33A, K53F, A58V, and V72Y. The superposition of Cab and P. sativum beta -CA structures clearly shows the distinctions in active site organization by these residues. His23, found on a flexible loop, is ~25 Å away from the catalytic zinc and probably is not a part of the active site. On the other hand, the equivalent residue Gln151 in the P. sativum beta -CA structure occupies a position similar to the Hepes sulfate group that is ~8 Å away from the zinc atom and is possibly involved in ligand binding (23). The hydrophobic pocket of Cab is quite different from that of plant-type beta -CA (Fig. 6A). In P. sativum beta -CA structure, the hydrophobic pocket is formed by Phe179, Val184, and Tyr205. The corresponding residues in Cab (Lys53, Ala58, and Val72) constitute a more open and less hydrophobic pocket.


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Fig. 6.   Stereo diagram showing the superposition of the active site of Cab, P. sativum, and P. purpureum beta -CAs including zinc, zinc-coordinating residues, and the conserved residues differentiating between cab- and plant-type beta -CA. A, Cab is shown in yellow; P. sativum beta -CA is shown in green. The coordinating water molecule in Cab is shown in red. B, Cab is shown in yellow; P. purpureum beta -CA is shown in blue. The coordinating water molecule in Cab is shown in red.

Comparison of P. purpureum beta -CA and Cab Active Sites-- Again, the protein ligands to the zinc in the two structures superpose closely (Fig. 6B). The fourth ligand is different, since the side chain of Asp151 in the P. purpureum beta -CA structure coordinates the zinc instead of the water molecule seen in Cab structure. Asp151 of P. purpureum beta -CA is equivalent to Asp34 of Cab. As a consequence of the zinc coordination by Asp151 in the P. purpureum beta -CA structure, this residue cannot pair with the conserved Arg, and Arg153 has flipped away from the active site. The adjacent Ser152 has moved by ~4 Å and adopts a different conformation in the P. purpureum beta -CA structure (Fig. 6B). The hydrophobic pocket arrangement of P. purpureum beta -CA is very similar to that of P. sativum beta -CA, and the differences between the cab-type and plant-type hydrophobic pocket have already been discussed.

Mechanism-- The catalytic mechanism of carbonic anhydrases has been most extensively studied for the alpha -CA class (5, 6, 40-42). The zinc hydroxide mechanism established for this class provides an appropriate framework for discussing the catalytic mechanism of Cab. In the first part of the CO2 hydration reaction, CO2 binds in the hydrophobic pocket and probably interacts with the amide nitrogen of Thr199. This threonine is known as the "gatekeeper," and the side chain plays an important role in the alpha -CAs, together with Glu106, in orienting the CO2 molecule for attack by the zinc-bound hydroxide. In the P. sativum beta -CA structure, Asp162, Gly224, and Gln151 are thought to play the same role in orienting CO2 for this attack (23). In Cab, Asp34 and Gly91 are in the same orientation as Asp162 and Gly224 in P. sativum beta -CA structure and might also help to orient CO2. His23, the equivalent of P. sativum beta -CA Gln151, is, however, disordered in active sites A, C, and E, and in active sites B, D, and F it is at the beginning of a segment of residues that pack against the symmetry-related molecule and lies 25 Å away from the active site zinc.

The second, rate-limiting, step in the CO2 hydration reaction involves the regeneration of a hydroxide ion from the zinc-bound water molecule. In alpha -CAII, the zinc ion is located in a deep funnel and requires a proton shuttle to transfer the proton to the bulk solvent. His64 of alpha -CAII adopts multiple conformations, which facilitates accepting the proton from the zinc-bound water molecule and delivering it to buffer in bulk solution (43). In gamma -CA, Glu84 exhibits multiple conformations and has been proposed to participate in a proton shuttle (18, 44). Residues with multiple conformations have not been described in the active site of any beta -CA structure determined so far. Since the beta -CA active site is closer to the surface of the protein than the alpha -CA active site, a protein-mediated proton shuttle might not be necessary. The beta -CA reaction rate depends on buffer concentration, implying that proton transfer can be rate-limiting under certain conditions (21). In the Cab structure, a Hepes buffer molecule was found near the active sites A, C, and E. The Hepes sulfate group is located ~8 Å away from the zinc atom and lies within hydrogen bonding distance of Asp34, which makes a hydrogen bond to the zinc bound water molecule. In the P. purpureum beta -CA structure, the equivalent of Asp34 acts as the fourth zinc ligand, and in the proposed mechanism it plays a role in the proton transfer (24). Therefore, the most plausible pathway for proton transfer in Cab is from the zinc-bound water molecule to Asp34 and then to the sulfate group of the bound Hepes molecule or a solvent molecule. The conformation of residues 1-25 in active sites B, D, and F is incompatible with Hepes binding, and residues 13-25 must adopt different conformations for Hepes to bind (Fig. 5B). The mobility of residues 1-25 and 92-125 might allow buffer molecules to diffuse into the active site and serve as the proton acceptor necessary to regenerate the zinc-bound hydroxide.

    ACKNOWLEDGEMENTS

We thank Jessica Wuu and Radu Georgescu for help with crystallizations and Brian R. Crane and Alex M. Bilwes for help with data collection.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM44661 (to J. G. F) and GM45162 (to D. C. R.); a National Science Foundation predoctoral fellowship (to P. S.); and NASA-AMES Cooperative Agreement NCC2-1057 (to the Pennsylvania State University Astrobiology Research Center). This work is based upon research conducted at the Stanford Synchrotron Radiation Laboratory, which is funded by the Department of Energy (Office of Basic Energy Sciences and Office of Biological and Environmental Research) and the National Institutes of Health (National Center for Research Resources, NIGMS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1G5C) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

** To whom correspondence should be addressed: Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 147-75CH, Pasadena, CA 91125. Tel.: 626-395-8393; Fax: 626-744-9524; E-mail: dcrees@caltech.edu.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009182200

    ABBREVIATIONS

The abbreviations used are: CA, carbonic anhydrase; Cab, M. thermoautotrophicum beta  class carbonic anhydrase; beta -CA, beta class carbonic anhydrase; r.m.s., root mean square; NCS, noncrystallographic symmetry.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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