Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal {beta}-Class Carbonic Anhydrase

The roles of an aspartate and an arginine, which are completelyconserved in the active sites of ß-class carbonicanhydrases, were investigated by steady-state kinetic analysesof replacement variants of the ß-class enzyme (Cab)from the archaeon Methanobacterium thermoautotrophicum. Previouskinetic analyses of wild-type Cab indicated a two-step zinc-hydroxidemechanism of catalysis in which the k_cat/K_m value depends onlyon the rate constants for the CO₂ hydration step, whereas k_catalso depends on rate constants from the proton transfer step(K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, andJ. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recentlysolved crystal structure of Cab shows the presence of a buffermolecule within hydrogen bonding distance of Asp-34, implyinga role for this residue in the proton transport step (P. Strop,K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J.Biol. Chem. 276:10299-10305, 2001). The k_cat/K_m values of Asp-34variants were decreased relative to those of the wild type,although not to an extent which supports an essential role forthis residue in the CO₂ hydration step. Parallel decreases ink_cat and k_cat/K_m values for the variants precluded any conclusionsregarding a role for Asp-34 in the proton transfer step; however,the k_cat of the D34A variant was chemically rescued by replacementof 2-(N-morpholino)propanesulfonic acid buffer with imidazoleat pH 7.2, supporting a role for the conserved aspartate inthe proton transfer step. The crystal structure of Cab alsoshows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variantshad both k_cat and k_cat/K_m values that were decreased at least250-fold relative to those of the wild type, establishing anessential function for this residue. Imidazole was unable torescue the k_cat of the R36A variant; however, partial rescueof the kinetic parameter was obtained with guanidine-HCl indicatingthat the guanido group of this residue is important.

INTRODUCTION

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Introduction

Carbonic anhydrase is a zinc-containing enzyme that catalyzesthe reversible hydration of carbon dioxide:

((1))
Thisenzyme, which is present in species from all three domains oflife, plays a critical role in many diverse physiological processessuch as respiration, photosynthesis, and CO₂ fixation.

Based on amino acid sequence comparisons, carbonic anhydrasesbelong to four genetically distinct classes ( ${alpha}$ , ß, ${gamma}$ , and ${delta}$ ) of independent origins (38). The crystal structuresfor representatives of the ${alpha}$ , ß, and ${gamma}$ classes havenow been determined (3, 7, 9-13, 16, 18, 19, 23, 26, 35, 36).Although the structure of the recently identified ${delta}$ -class prototypefrom the diatom Thalassiosira weissflogii has not yet been solved,extended X-ray absorption fine-structure analyses suggest thatthe active-site zinc is coordinated by three histidines andone water molecule, as found in the ${alpha}$ and ${gamma}$ classes (6). The structuresof the ß-class carbonic anhydrases (7, 18, 26, 36)indicate striking differences between this class and the others.For example, the active-site zinc in the ß-class enzymesis coordinated by two cysteines, one histidine, and one watermolecule.

The kinetic properties of the human ${alpha}$ -class isozymes have beencomprehensively investigated and follow a common zinc-hydroxidemechanism for catalysis (24, 30). The catalytically active groupin this mechanistic model is the zinc-bound water, which ionizesto a metal-bound hydroxyl which attacks CO₂. Despite considerablestructural differences in the active sites of these enzymes,the catalytic mechanisms of both ${gamma}$ - and ß-class enzymesresemble those of human ${alpha}$ -class HCA II (1, 14, 15, 28, 31). Theoverall enzyme-catalyzed reaction occurs in two mechanisticallydistinct steps (where E = enzyme and B = buffer):

((2a))

((2b))

((2c))

((2d))

The first step is the interconversion between carbon dioxideand bicarbonate (equations 2a and 2b) and involves the nucleophilicattack of the zinc-bound hydroxyl on the CO₂ molecule. In the ${alpha}$ -class enzyme, the zinc-bound oxygen forms a hydrogen bond withThr-199 (22, 42, 43). The role of this conserved gatekeeperresidue is to prevent nonprotonated atoms from binding effectively.In addition, Thr-199 has been proposed to electrophilicallyactivate the CO₂ molecule by forming a hydrogen bond with CO₂through its backbone amide. The second step of the proposedmechanism is the regeneration of the zinc hydroxide at the activesite of the enzyme (equations 2c and 2d), involving proton transferevents. The k_cat/K_m value depends only on the rate constantsfor the CO₂ hydration steps (equations 2a and 2b), whereas k_catalso depends on rate constants from the proton transfer steps(equations 2c and 2d). Carbonic anhydrases with slower rates(k_cat of less than 10⁴ s^-1) transfer the proton directly tobuffer or water molecules in solution, as this is the fastestrate at which protons can transfer from an acidic group witha pK_a of 7 to water (25). Faster carbonic anhydrases with turnovernumbers in the range of 10⁴ to 10⁶ s^-1 transfer the proton fromthe metal-bound water molecule to an intermediate proton shuttleresidue and then to an external buffer molecule. In the ${alpha}$ -classHCAII, the proton first undergoes intramolecular transfer tothe proton shuttle residue His-64 (40). In the ${gamma}$ -class enzymeCam, this proton transfer is to Glu-84 (37). The proton is subsequentlytransferred to an accepting buffer molecule in the surroundingmedia during an intermolecular proton transfer step. Imidazoleis able to rescue the k_cat of variant carbonic anhydrases inwhich the proton shuttle residue is replaced with alanine, presumablyby entering the active site and replacing the function of theproton shuttle residue.

Previously, the lack of a structure for a ß-classcarbonic anhydrase hindered work on this class relative to thatof the ${alpha}$ class and even the recently discovered ${gamma}$ class. However,the structures of four ß-class enzymes have been reportedin the last 2 years (7, 18, 26, 36). Comparison of these structures,in addition to phylogenetic analysis, indicates that the ßclass is composed of two subclasses (34, 38). The plant-typesubclass is represented by the Pisum sativum (pea) enzyme andis composed of enzymes from the Eucarya domain and gram-negativespecies in the Bacteria domain. The other subclass, the cab-type,is represented by the enzyme Cab from the thermophilic methane-producingarchaeon Methanobacterium thermoautotrophicum. The cab-typesubclass includes carbonic anhydrases from the Archaea domainand gram-positive species from the Bacteria domain.

Little is known concerning what residues play a critical rolein the zinc hydroxide mechanism for the ß-class carbonicanhydrases compared with the ${alpha}$ and ${gamma}$ classes. Only five residuesare completely conserved in the diverse ß class (33,34). Three of these residues (two cysteines and one histidine)chelate the active-site zinc. Roles for the other two conservedresidues, an aspartate and an arginine, have not been determined.A variant of the Spinacia oleracea enzyme in which the conservedaspartate was replaced (D152N) retained approximately 1% ofthe specific activity of the wild-type enzyme; however, no kineticanalyses were reported (4). Therefore, the role of the conservedaspartate in the ß-class enzymes remains unclear.Here, we report the first kinetic characterization of Cab variantswith replacements at the conserved aspartate (Asp-34) and arginine(Arg-36).

MATERIALS AND METHODS

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Materials and Methods

Enzyme purification. The wild-type and variant carbonic anhydrases were heterologouslyproduced in Escherichia coli BL21(DE3) (Novagen, Inc.) and purifiedas previously described (31, 32). Enzyme activity during purificationwas measured at room temperature by using a modification ofthe electrometric method of Wilbur and Anderson (41). Proteinconcentrations were determined by the Bradford method with Bio-Raddye reagent and bovine serum albumin (Sigma) as the standard(5).

Site-directed mutagenesis. Mutagenesis was performed by oligonucleotide-directed in vitromutagenesis by using the QuikChange kit (Stratagene) accordingto the manufacturer's instructions. Plasmid pMBTCA13 (32), aderivative of the plasmid pET15a (Novagen, Inc.) containingthe M. thermoautotrophicum cab gene, was the target plasmidfor site-directed mutagenesis. The mutations were confirmedby dye termination cycle sequencing with an ABI PRISM 377 DNAsequencer (Perkin Elmer Life Sciences) at the Nucleic Acid Facilityat Pennsylvania State University.

Steady-state kinetics. Initial rates of CO₂ hydration were determined by stopped-flowspectroscopy (KinTek stopped-flow apparatus; KinTek, State College,Pa.) at 25°C by using the changing pH indicator method (17).Saturated solutions of CO₂ (32.9 mM) were prepared by bubblingCO₂ into distilled deionized water at 25°C, and the finalCO₂ concentrations (6 to 24 mM) were obtained by dilution withN₂-saturated buffer. The following buffer and pH indicator pairs(and wavelengths) were used: at pH 6.4 and 6.8, MES [2-(N-morpolino)ethanesulfonicacid] (pK_a = 6.1) and chlorophenol red (574 nm); at pH 7.2,MOPS [2-(N-morpholino)propanesulfonic acid] (pK_a = 7.2) andp-nitrophenol (400 nm); at pH 7.2, imidazole (pK_a = 6.9) andp-nitrophenol (400 nm); at pH 7.6, HEPES (pK_a = 7.5) and phenolred (557 nm); at pH 8.0 to 9.0, TAPS [N-tris(hydroxymethyl)methyl-3-aminopropanesulfonicacid] (pK_a = 8.4) and m-cresol purple (578 nm). The observedinitial rates were corrected for the uncatalyzed rate of thereaction, which was at least five times lower than the catalyzedrate. The steady-state parameters k_cat and k_cat/K_m and theirstandard errors were then determined by fitting the observedinitial rates to the Michaelis-Menten equation. All fits describedwere performed by using KaleidaGraph (Synergy Software, Reading,Pa.).

Materials. Chemicals were purchased from Sigma, VWR Scientific, or Fisher.Oligonucleotide primers for site-directed mutagenesis were obtainedfrom Integrated DNA Technologies, Inc.

RESULTS

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Results

Initial characterization of Asp-34 and Arg-36 replacement variants of Cab. The roles of the two completely conserved residues in the ßclass (excluding zinc ligands) were examined in Cab to beginto understand the catalytic mechanism of ß-class carbonicanhydrases. Thus, Asp-34 and Arg-36 of Cab were replaced bysite-directed mutagenesis (Table 1), and the variant enzymeswere kinetically characterized. Gel filtration chromatographyindicated that the variants are all tetramers, in accordancewith the wild-type enzyme. In addition, all the variants exhibitedpatterns of temperature stability similar to those of the wild-type(data not shown). These results indicate that the variants experiencedno gross structural perturbations.

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TABLE 1. Steady-state kinetic parameters for wild-type Cab and Cab variants with TAPS buffer (pH 8.5)^a

Kinetic analysis of Asp-34 and Arg-36 replacement variants of Cab. The variants were analyzed by stopped-flow spectroscopy in thedirection of CO₂ hydration. As previously shown for wild-typeCab (31), the assay progress curves for all the variants wereconsistent with Michaelis-Menten kinetics (data not shown).The k_cat or k_cat/K_m values for both Asp-34 replacement variantsdecreased no more than 10-fold relative to those of the wildtype when assayed with either MOPS or TAPS buffer (Tables 1and 2); however, both of the Arg-36 replacement variants hadk_cat or k_cat/K_m values that were reduced between 250- and 6,000-foldcompared to those of the wild type. The D36K variant had k_catand k_cat/K_m values greater than those of the D36A variant. Analysisof the D34A-R36A double variant revealed steady-state parametersthat were intermediate to those of the D34A and R36A variants.

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TABLE 2. Steady-state kinetic parameters for wild-type Cab and Cab variants with MOPS and imidazole buffers (pH 7.2)

The k_cat and k_cat/K_m values for wild-type Cab and all of thevariants were pH dependent between pH 6.4 and 9.0 and increasedwith increasing pH (Fig. 1). These results indicate that thefundamental zinc hydroxide mechanism is operable for the variants,as determined previously for wild-type Cab (31). As previouslyreported for wild-type Cab (31), the pH profiles of either steady-stateparameter for the variants could not be fit to a theoreticaltitration curve with one, two, or three ionizations.

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FIG. 1. pH-dependent steady-state kinetic parameters for wild-type Cab and Cab variants. CO₂ hydration was measured at 25°C in 50 mM buffer with an ionic strength of 0.1 M. The observed steady-state parameters were determined over a pH range of 6.4 to 9.0. (A) k_cat values for wild-type (filled circles), D34A (filled squares), R36A (open circles), and D34A-R36A (open squares) enzymes are shown. (B) k_cat/K_m values for wild-type (filled circles), D34A (filled squares), R36A (open circles), and D34A-R36A (open squares) enzymes are shown.

Imidazole rescue of Cab variants. The ability of imidazole to rescue k_cat for both ${alpha}$ - and ${gamma}$ -classcarbonic anhydrase variants is considered strong experimentalevidence for involvement of the replaced residue in the intramolecularproton transfer step of catalysis. Thus, the k_cat values forwild-type Cab and the variants were determined in the presenceof either imidazole or MOPS buffer at pH 7.2 (Table 2). Thek_cat values for the wild type and the R36A variant were notsignificantly altered with either buffer. However, the k_catvalues for the D34A and D34A-R36A variants were eight- and sixfoldgreater in the presence of imidazole than in the presence ofMOPS. The k_cat value for the D34A variant was also dependenton the imidazole concentration, with k_cat approaching a maximumvalue at approximately 150 mM (Fig. 2A). The k_cat values forthe wild type and the R36A variant were independent of the concentration(0 to 200 mM) of imidazole (Fig. 2A). Replacing MOPS with imidazoledid not significantly change the k_cat/K_m value for the wildtype or the R36A variant (Table 2). However, the k_cat/K_m valuefor the D34A variant decreased nearly 5-fold, whereas the kineticparameter for the D34A-R36A variant increased nearly 10-foldwith imidazole compared to MOPS. The k_cat/K_m value for the wildtype or the variants was independent of the concentration (25to 200 mM) of imidazole (Fig. 2B).

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FIG. 2. Dependence on imidazole concentration of steady-state kinetic parameters for wild-type Cab and Cab variants. CO₂ hydration was measured at 25°C with an ionic strength of 0.1 M and buffer concentrations of 20 to 200 mM imidazole, pH 7.2. (A) k_cat values for wild-type (filled circles), D34A (filled squares), R36A (open circles), and D34A-R36A (open squares) enzymes are shown. (B) k_cat/K_m values for wild-type (filled circles), D34A (filled squares), R36A (open circles), and D34A-R36A (open squares) enzymes are shown.

Guanidine-HCl rescue of Cab variants. Replacement of the conserved Arg-36 had a substantial effecton the k_cat and k_cat/K_m values of Cab, suggesting that the guanidoside chain of Arg-36 may be important for catalysis. The steady-stateparameters of the R36A variant were examined in the presenceof increasing concentrations of guanidine-HCl (Fig. 3) to addressthe function of this residue. The k_cat values of the wild-typeCab remained constant between 0 and 400 mM guanidine-HCl, whereasthe k_cat of the R36A variant increased between 0 and 200 mM.However, guanidine-HCl concentrations greater than 400 mM decreasedthe k_cat values for both the wild type and the variant. Theeffect of guanidine-HCl on the k_cat value of the R36K variantwas similar to that of the wild-type Cab (data not shown). Concentrationsof 100 to 1,000 mM urea or methylamine had no significant effecton the k_cat of either Arg-36 replacement variant (data not shown).

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FIG. 3. Effect of guanidine-HCl on the steady-state parameters of the wild type and the R36A variant. CO₂ hydration was measured at 25°C in 50 mM TAPS (pH 8.5) with an ionic strength of 0.1 M in the presence of various concentrations of guanidine-HCl. k_cat values for wild-type (filled squares) and R36A (open squares) enzymes are shown.

DISCUSSION

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Discussion

Although the ß-class carbonic anhydrases appear tobe the most widely distributed in nature and the only classto have been documented in all three domains (33, 38), lessis known about the biochemistry of this class than about eitherof the other two classes. Of the ß-class carbonicanhydrases, only two plant-type ß-class carbonic anhydrasesand one cab-type ß-class carbonic anhydrase have beenkinetically characterized (14, 15, 28, 31). Even before thestructure of any ß-class enzyme had been determined,Asp-34 and Arg-36 of Cab were obvious targets for site-specificreplacement to determine their roles in catalysis. They representthe only residues that are completely conserved in all enzymesof the ß class, aside from the two cysteines and onehistidine which chelate zinc (33). A variant of the S. oleraceaenzyme, in which the conserved aspartate (Asp-152) was replacedwith asparagine (4), had 1% of the activity of the wild typewhen assayed in 25 mM EPPS buffer (4) at pH 8.0 and 9% of theactivity of the wild type when assayed in 25 mM imidazole bufferat pH 8.0. These results are consistent with a role for Asp-152as a proton shuttle residue; however, a kinetic analysis ofthe D152N variant has not been reported to further investigatethis proposed role. No studies are reported which address therole of the conserved aspartate in any of the ß-classcarbonic anhydrases for which the crystal structure is known.Thus, the role of the conserved aspartate in the catalytic mechanismfor any of the ß-class carbonic anhydrases has notbeen resolved. Based on the crystal structure, it was proposedthat one possible function for the Asp-34 of Cab is to orientCO₂ for attack by the zinc-bound hydroxyl (36). The replacementof Asp-34 in Cab with either alanine or glutamate produced variantswith modest decreases in k_cat/K_m values relative to those ofthe wild-type enzyme, suggesting that Asp-34 has no essentialrole in the CO₂ hydration step of catalysis. The crystal structureof Cab complexed with a HEPES buffer molecule is also consistentwith a role for Asp-34 as a proton shuttle residue (36). Asp-34forms a hydrogen bond to the zinc-bound water molecule, anda HEPES molecule is found approximately 8 Å from the catalyticzinc, within hydrogen bonding distance of Asp-34 (Fig. 4). Therefore,one possible pathway for proton transfer in the cab-type ß-classenzymes is from the zinc-bound water molecule to Asp-34 andthen to HEPES. The solvent hydrogen isotope effect on k_cat (2.1± 0.1) for wild-type Cab suggests that an intramolecularproton transfer step is at least partially rate determining(31); however, comparable decreases in both k_cat and k_cat/K_mfor the Asp-34 replacement variants preclude any conclusionsfrom the kinetic analyses regarding a proton transfer role forthis residue in catalysis. Nonetheless, imidazole rescue ofk_cat for the D34A variant and the D34A-R36A double variant suggestsa role for Asp-34 in proton transfer. A route for proton transfercould not be discerned from the crystal structures of the Porphyridiumpurpureum, P. sativum, or E. coli ß-class enzymes(7, 18, 26).

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FIG. 4. View of the Cab active site complexed with HEPES. The active site is shown with the three conserved zinc ligands Cys-32, His-87, and Cys-90 in yellow, the catalytic zinc in red, the zinc-bound water in magenta, the conserved Asp-34 in green, and the putative proton-accepting HEPES buffer molecule in blue. Hydrogen bonds are indicated by broken lines. The image was prepared with Web Lab Viewer.

In Cab, the conserved aspartate (Asp-34) is held in place bya pair of hydrogen bonds from Arg-36, the only other residuecompletely conserved among the ß-class sequences (36)(Fig. 5). Replacements of the conserved arginine in Cab producedvariants with substantial decreases in both k_cat and k_cat/K_mvalues relative to those of the wild type. Replacement of Arg-36with lysine had a similar effect as replacement with alanine,suggesting that, in addition to the positive charge at thisposition, the two-point interaction of the guanido group withAsp-34 is important. Chemical rescue of kinetic parameters withguanido derivatives has been reported for arginine replacementvariants of several enzymes (2, 8, 20, 21, 27, 29), includingCam, the prototype of the ${gamma}$ -class carbonic anhydrase (39). Thesederivatives are thought to occupy the cavity vacated by thearginine side chain in a productive conformation which mimicsthe guanido group of arginine. Guanidine-HCl rescued the k_catfor the Arg-36 replacement variants of Cab. The inability ofurea to rescue the variants argues against a role for guanidine-HClin rescuing the variants simply by restoring the conformationof the active site. The inability of methylamine to rescue thevariants indicates that a positive charge alone is not sufficientfor rescue. The results suggest that guanidine-HCl diffusesinto the active site of the variants to mimic the guanido groupof Arg-36. Higher guanidine-HCl concentrations (above 400 mM)resulted in a large decrease in k_cat and k_cat/K_m values forboth the R36A variant and the wild-type enzyme consistent withdenaturation.

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FIG. 5. View of the Cab active site showing the hydrogen bonding between Arg-36 and Asp-34. The active site is shown with the three conserved zinc ligands Cys-32, His-87, and Cys-90 in yellow, the catalytic zinc in red, the zinc-bound water in magenta, and the conserved Asp-34 and Arg-36 in green. Hydrogen bonds are indicated by broken lines. The image was prepared with Web Lab Viewer.

An explanation for why replacement of Arg-36 decreased the kineticparameters of Cab may come from inspection of the duplicatedactive sites in the crystal structure of the P. purpureum enzyme(26). The conserved arginine does not form hydrogen bonds tothe conserved aspartate in either of the duplicated active sites;instead, the aspartates coordinate to zinc while the arginineside chains are flipped away from the active sites. This structureled the authors to propose a modified zinc hydroxide mechanismin which the aspartate ligand to zinc is exchanged with a hydroxylduring catalysis. Aspartate ligation to zinc may be occurringin the Arg-36 replacement variants of Cab where the hydrogenbonds to Asp-34 would be disrupted. In the R36A variant, Asp-34may swing toward the active site and ligate zinc in a dead-endinactive conformation. If this hypothesis is correct, a variantin which both Asp-34 and Arg-36 are replaced with alanine wouldbe expected to have k_cat and k_cat/K_m values greater than thoseof the R36A variant since Asp-34 could no longer ligate zincin the double variant. Indeed, the k_cat and k_cat/K_m values forthe D34A-R36A variant in TAPS buffer were 11- and 430-fold greaterthan those for the R36A variant. These results are consistentwith the function of Arg-36 to hold Asp-34 in the active sitethrough two hydrogen bonds; however, an exchange of ligandsto Asp-34 during catalysis cannot be ruled out. Indeed, an exchangeof ligands may be necessary if the pK_a of Asp-34 were decreasedwhen hydrogen bonded to Arg-36 such that Asp-34 is unable toabstract a proton from the zinc-bound water. Another potentialfunction for Arg-36 may be to bind bicarbonate after releasefrom zinc, thereby facilitating product removal, as proposedfor arginine in the active site of Cam (39).

ACKNOWLEDGMENTS

We thank Matthew Kimber for invaluable discussions regardingthe active sites of ß-class carbonic anhydrases.

This work was supported by grants from the National Institutesof Health to J.G.F. (no. GM44661) and from the NASA-Ames CooperativeAgreement NCC2-1057 to The Pennsylvania State University AstrobiologyResearch Center.

FOOTNOTES

* Corresponding author. Mailing address: Center for Microbial Structural Biology, Department of Biochemistry and Molecular Biology, 205 South Frear Laboratory, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-5721. Fax: (814) 863-6217. E-mail: jgf3{at}psu.edu.

REFERENCES

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