Role of Hydrophilic Residues in Proton Transfer During Catalysis by Human Carbonic Anhydrase II ^,

Abstract

Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO₂ for the resulting mutants has been characterized by ¹⁸O exchange, and the structures of the mutants determined by X-ray crystallography to 1.5 – 1.7 Å resolution. Each of these mutants maintained the ordered water structure observed by x-ray crystallography in the active-site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, V, L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in pK_a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active-site cavity in maintaining efficient catalysis by carbonic anhydrase.

The carbonic anhydrases comprise several well studied classes among which is the α class including the human and animal enzymes (1, 2). The zinc metalloenzyme human carbonic anhydrase II (HCA II) is the most extensively studied of these enzymes and catalyzes the hydration of CO₂ by a two stage mechanism (3-5). The first is the reaction of CO₂ with zinc-bound hydroxide to form and dissociate bicarbonate (eq 1). The second is the regeneration of the zinc-bound hydroxide by proton transfer to solution (eq 2).

$${\mathrm{CO}}_2+{\mathrm{EZnOH}}^{-}\rightleftarrows {{\mathrm{EZnHCO}}_3}^{-}\stackrel{{\mathrm{H}}_2\mathrm{O}}{\rightleftarrows }{\mathrm{EZnH}}_2\mathrm{O}+{{\mathrm{HCO}}_3}^{-}$$ (1)

$$\mathrm{His}64-{\mathrm{EZnH}}_2\mathrm{O}+\mathrm{B}\rightleftarrows {\mathrm{H}}^{+}\mathrm{His}64-{\mathrm{EZnOH}}^{-}+\mathrm{B}\rightleftarrows \mathrm{His}64-\mathrm{EZn}{\mathrm{OH}}^{-}+{\mathrm{BH}}^{+}$$ (2)

Here B is an exogenous proton acceptor and His64 is the internal proton shuttle residue (6). The rate limiting step in maximal velocity of this catalysis is the transfer of a proton between His64 and the zinc-bound solvent (6, 7). His64 is located on the rim of the active-site cavity with its side chain extending into the cavity (Figure 1)(5, 8, 9). In crystal structures two orientations are observed for this side chain (9, 10), one is an inward conformation with the side chain oriented towards the zinc, and a second is an outward conformation with His64 oriented towards the mouth of the active site cavity and external solution.

Figure 1.

The active site of HCA II from the data of Fisher et al. (38). The side chain of His64 is shown in both the inward and outward conformations. The red spheres represent oxygen including the oxygens of ordered water molecules numbered W1, W2, W3a, and W3b. Dashed red lines indicate presumed hydrogen bonds. This figure was generated and rendered with PyMOL (www.pymol.org).

The Nδ1 of the imidazole side chain of His64 in HCA II in the inward conformation is too far from the zinc (about 7.5 Å; (10, 11)) for direct proton transfer, and solvent hydrogen isotope effects are consistent with proton transfer through intervening hydrogen-bonded solvent bridges (12). Crystal structures of the isozymes of CA in the α class exhibit networks of ordered, apparently hydrogen-bonded water molecules between His64 and the zinc-bound water in the active site cavity (Figure 1). In the Grotthuss mechanism, high proton mobility is achieved by proton shuttling along hydrogen bonds without requiring much motion from their oxygen atoms. However, the apparently hydrogen bonded water structure shows at best weak hydrogen bonding between the Nδ1 atom of the side chain of His64 (inward orientation) and the oxygen atom of the nearest water molecule W2, a distance which is 3.2 (Figure 1) (11). The proton migration in CA is of great current interest as a model for proton transfer in more complex systems, and is also under study in several labs using computational methods (13-17). These continuing studies of CA are significant in understanding the role of water in facilitating proton transfers in proteins and in catalysis.

Recent attention has focused on the significance of the ordered solvent observed in the crystal structures of CA. Several residues have been shown to stabilize this ordered solvent structure, among them Tyr7, Asn62, and Asn67 (Figure 1)(18, 19). Fisher et al. (18) replaced these hydrophilic residues with hydrophobic residues (Y7F, N62L, and N67L) and observed several changes in structure and catalysis. These mutations had different effects on the orientation of the side chain of His64 (Y7F and N62L predominantly inward and N67L predominantly outward), altered the pK_a of His64, and affected the ordered water structure. The observed rate constants for proton transfer between His64 and the zinc-bound hydroxide were changed for these mutants, but there was little effect on the rate constant for conversion of CO₂ into bicarbonate (18). Among these the mutant N62L was interesting since the X-ray crystal structure showed His64 in the inward orientation and the ordered water structure intact as in the wild type (18).

The side chain of Asn62 in HCA II is extended into the active-site cavity about 9 Å from the zinc and as close at 3.3 Å from the side chain of His64 (Figure 1)(8-10). This residue appears conserved in many species of CA II, for example in mouse and chicken as well as in many other isozymes of carbonic anhydrase (20). Computations of conformational states of active-site residues in HCA II point out steric interactions between the side chains of His64 and Asn62 that contribute to the orientation of His64 (17, 19). In previous experimental studies, the pH profile of catalysis by N62L HCA II appeared irregular and difficult to interpret (18). However, additional amino acid substitutions at position 62 in this study have provided a more straightforward interpretation. The replacement of Asn62 with other amino acids was shown to cause changes in the orientation of the proton shuttle His64 and the pK_a of its imidazole side chain, but had little or no effect on the structure of ordered solvent in the active-site cavity. The results point out the capacity of His64 to participate in proton transfer more efficiently in the inward than in the outward conformations, and the role of residue 62 in fine tuning the values of pK_a of His64 and the zinc-bound solvent molecule.

MATERIALS AND METHODS

Expression and Purification of HCA II mutants

The mutants N62D, N62A, N62V and N62T of HCA II were made by site-directed mutagenesis using an expression vector containing wild type HCA II coding region (6). The point mutations were made using the QuikChange II Kit (Stratagene, LaJolla) for site-directed mutagenesis. The single mutants were confirmed by DNA sequencing over the entire coding region of HCA II. Expression of each mutant was done by transforming mutated vectors into Escherichia coli BL21(DE3)pLysS cells, which do not express any indigenous CA under the following conditions. The transformed cells were expressed at 37 °C in LB medium containing 100 μg/ml ampicillin. HCA II production was induced by the addition of isopropyl thiogalactoside to a final concentration of 1mM when the bacterial culture reached an OD₆₀₀ of 0.6. The cells were harvested 4 hrs after induction. The cell pellets were lysed and HCA II was purified through affinity chromatography using p-(aminomethyl)benzenesulfonamide coupled to agarose beads (21). Electrophoresis on a 10% polyacrylamide gel stained with Coomassie Blue was used to confirm the purity of enzyme samples, which were found to be greater than 96% pure. HCA II and the mutants studied here bound sulfonamides tightly; therefore, enzyme concentrations were determined by titration of active sites with ethoxzolamide while measuring the catalyzed depletion of ¹⁸O from CO₂ and analyzing data with the Henderson approach (22).

Crystallography

Crystals of the HCAII single-site mutants of N62A, N62V, N62T and N62D were obtained using the hanging-drop vapor diffusion method (23). The crystallization drops were prepared by mixing 5 μL of protein (10mg/mL) in 50 mM Tris-HCl (pH 7.0) with 5 μL of the precipitant solution 50 mM Tris-HCl (pH 9.0) and 1.3 M sodium citrate at 20 °C against 1 ml of the precipitant solution. The pH of the crystallization solutions was about 7.9. Useful crystals were observed 4 days after the crystallization setup. Previous studies had shown the binding of sulfate at the zinc in N62L HCA II (18); hence sulfate was not used as a precipitant in crystallizations reported here. The X-ray diffraction datasets for all of the mutant HCAII crystals were obtained at room temperature, using an R-AXIS IV⁺⁺ image plate system with Osmic mirrors and a Rigaku RU-H3R Cu rotating anode operating at 50 kV and 100 mA. The detector-crystal distance was set to 80 mm. Each dataset was collected at room temperature with the crystals mounted in quartz capillaries. The oscillation steps were 1° with a 7 min exposure per image. X-ray data processing was performed using DENZO and scaled and reduced with SCALEPACK (24). All manual model building was performed using Coot (25) and refinement was carried out with the crystallography and nuclear magnetic resonance system (CNS) suite of programs, version 1.1 (26).

The wild-type HCA II crystal space group [Protein Data Bank (PDB) accession code 2CBA (11)] was isomorphous with all of the datasets collected, and was used to phase the datasets. To avoid phase bias of the model, the zinc ion, mutated residues, and water molecules were removed. After one cycle of rigid-body refinement, annealing by heating to 3000 K with gradual cooling, geometry-restrained position refinement, and temperature-factor refinement, 2Fo - Fc and Fo - Fc Fourier electron-density maps were generated. These electron-density maps clearly showed the position of the zinc and mutated residues, which were subsequently built into their respective models. After several cycles of refinement, solvent molecules were incorporated into the models using the automatic waterpicking program in CNS until no more water molecules were found at a 2.0σ level. Refinement of the models continued until convergence of R_cryst and R_free was reached (see Table 1).

Table 1.

Crystal structure data and refinement statistics of four variants of HCA II.

	N62A	N62V	N62T	N62D
unit cell dimensions, a, b, c (Å), β ( ° )	42.8 41.7 72.9 104.6	42.8 41.7 72.9 104.6	42.7 41.6 72.8 104.6	42.7 41.7 72.8 104.5
resolution (Å)	50 – 1.7 (1.76-1.7)	50 - 1.5 (1.55-1.5)	50 - 1.6 (1.66-1.6)	50 – 1.6 (1.66-1.6)
No. of unique reflections	25972(2500)a	37008(3472)	31194(2971)	31546(2992)
completeness (%)	93.8(91.5)	92.2(87.0)	94.3(90.6)	95.2(91.1)
redundancy	2.4(2.3)	2.6(2.5)	2.9(2.9)	2.9(2.7)
Rsymmb(%)	0.08(0.45)	0.074(0.42)	0.065(0.349)	0.054(0.33)
Rcrystc /Rfreed(%)	17.9 / 19.5	18.4 / 19.7	17.05 / 19.5	17.9 / 18.3
No of protein/solvent atoms	2055/110	2057/201	2057/179	2058/289
average B factors(Å2) main/side chain Zn/solvent	18.2/21.3 11.9/36.3	15.8/19.0 9.5/35.7	13.1/16.2 6.8/29.3	15.3/18.3 9.3/42.3
Ramachandran Plot (%). most favored/ additionally allowed,	96.9/3.1	96.9/3.1	96.1/3.9	96.5/3.5
eRMSD (Å)	0.089	0.092	0.08	0.087
I / σ(I)	25.3 (3.1)	31.3 (3.2)	12.9 (3.1)	28.8 (4.6)
R.m.s.d. for bond lengths (Å) / angles ( ° )	0.007 / 1.3	0.004 / 1.3	0.004 / 1.3	0.005 / 1.3
PDB accession number	3DV7	3DVB	3DVC	3DVD

^aValues in parenthesis are for the highest resolution shell.

^bR_symm = Σ |I - <I>| / Σ <I> ×100

^cR_cryst = Σ |Fo| — |Fc| / Σ |Fobs| × 100

^dR_free is calculated the same as R_cryst, except it uses 5% of reflection data omitted from refinement.

^eThe root mean square deviation of C_α positions as compared to the wild-type crystal structure of HCAII (PDB ID: 2CBA)(11).

Oxygen-18 Exchange

This method is based on the measurement by membrane inlet mass spectrometry of the depletion of ¹⁸O from species of CO₂ (27, 28). A continuous measure of isotopic content of CO₂ is provided by CO₂ passing across the membrane where it enters a mass spectrometer (Extrel EXM-200). In the first of two independent stages of catalysis, the dehydration of labeled bicarbonate has a probability of transiently labeling the active site with ¹⁸O (eq 3). In a second stage, the protonation of the zinc-bound ¹⁸O-labeled hydroxide results in the release of H₂¹⁸O to the solvent (eq 4)

$${\mathrm{HCOO}}^{18}{\mathrm{O}}^{-}+{\mathrm{EZnH}}_2\mathrm{O}\stackrel{-{\mathrm{H}}_2\mathrm{O}}{\rightleftarrows }{\mathrm{EZnHCOO}}^{18}{\mathrm{O}}^{-}\rightleftarrows \mathrm{COO}+{\mathrm{EZn}}^{18}{\mathrm{OH}}^{-}$$ (3)

$${\mathrm{H}}^{+}\mathrm{His}64-{\mathrm{EZn}}^{18}{\mathrm{OH}}^{-}\rightleftarrows \mathrm{His}64-{{\mathrm{EZnH}}_2}^{18}\mathrm{O}\stackrel{{\mathrm{H}}_2\mathrm{O}}{\rightleftarrows }\mathrm{His}64-{\mathrm{EZnH}}_2\mathrm{O}+{{\mathrm{H}}_2}^{18}\mathrm{O}$$ (4)

Two rates for the ¹⁸O exchange catalyzed by carbonic anhydrase are obtained by this method (27). The first is R₁, the rate of exchange of CO₂ and HCO₃^- at chemical equilibrium, as shown in eq 5. Here k_cat^ex is a rate constant for maximal interconversion of substrate and product, K_eff^S is an apparent binding constant for substrate to enzyme, and [S] is the concentration of substrate, either CO₂ or bicarbonate (29). The ratio k_cat^ex/ K_eff^CO2 is, in theory and in practice, equal to k_cat/K_m for hydration obtained by steady-state methods.

$${\mathrm{R}}_1∕\left[\mathrm{E}\right]={k_{\text{cat}}}^{\mathrm{ex}}\left[{\mathrm{CO}}_2\right]∕({{\mathrm{K}}_{\text{eff}}}^{\mathrm{CO}2}+[{\mathrm{CO}}_2\left]\right)$$ (5)

R_H2O, the rate of release from the enzyme of water bearing substrate oxygen (eq 4), is the second rate determined by the ¹⁸O-exchange method. This is the component of the ¹⁸O exchange that is dependent upon the donation of protons to the ¹⁸O-labeled zinc-bound hydroxide (6, 27). In such a step, His64 as a predominant proton donor in the catalysis provides a proton (eq 4). In eq 6, k_B is the rate constant for proton transfer to the zinc-bound hydroxide and (K_a)_donor and (K_a)_ZnH2O are the ionization constants of the proton donor and zinc-bound water molecule. The determination of the kinetic constant k_B and ionization constants of eq 6 was carried out by nonlinear least-squares methods (Enzifitter, Elsevier-Biosoft, Cambrige, U.K.)

$${{\mathrm{k}}_{\mathrm{B}}}^{\mathrm{obs}}=k_{\mathrm{B}}∕\left(\right[1+{\left(K_{\mathrm{a}}\right)}_{\text{donor}}∕\left[{\mathrm{H}}^{+}\right][1+[{\mathrm{H}}^{+}]∕{\left(K_{\mathrm{a}}\right)}_{\mathrm{Zn}\mathrm{H}2\mathrm{O}}])$$ (6)

The catalyzed and uncatalyzed exchange of ¹⁸O between CO₂ and water at chemical equilibrium were measured in the absence of buffer at a total substrate concentration of 25 mM using membrane-inlet mass spectrometry (27). The temperature was 25 °C and the total ionic strength of solution was kept at a minimum of 0.2 M by the addition of Na₂SO₄.

Esterase activity

The hydrolysis of 4-nitrophenylacetate uncatalyzed and catalyzed by variants of carbonic anhydrase was measured by the method of Verpoorte et al. (30). The increase in absorbance at 348 nm was measured; this is the isosbestic point of the product nitrophenol and the nitrophenolate anion with an absorbtivity of 5.0 × 10³ M^-1cm^-1. Initial velocities were measured using a Beckman Coulter DU 800 spectrophotometer.

RESULTS

We examined the crystal structures and catalytic properties of four mutants of HCA II containing substitutions at position 62, namely, N62A, D, V, and T. In addition, these properties for N62L were reported previously (18).

Crystallography

The crystals of the four mutant forms of HCA II were isomorphous with the wild type enzyme with mean unit cell dimensions of a = 42.7 ± 0.1 Å, b = 41.7 ± 0.1 Å, c = 72.8 ± 0.1 Å and β = 104.6 ± 0.1° (Table 1, Figures 2,3). All data sets were greater than 92% complete and were processed to 1.7 - 1.5 Å resolution (Table 1). A least squares superposition of these mutants with the crystal structure of wild-type HCA II (PDB ID: 2CBA; (11)) gave an average RMSD of Cα atoms 0.09 ±0.01 Å. The polypeptide backbone at position 62 was shifted slightly into the active-site cavity for the mutants N62A, N62V and N62T with a Cα movement of 0.6, 0.5, and 0.5 Å respectively.

Figure 2.

The active site of site-specific mutants (A) N62A, (B) N62V, (C) N62T, and (D) N62D HCA II. Residues are labeled and shown as stick models; the zinc atom is depicted as a white sphere and the oxygens of solvent water molecules as red spheres. Note the side chain of His64 is orientated inward for N62A and N62V, both inward and outward for N62T, and outward for N62D HCA II. The F_o-F_c electron density map was generated by omitting the residues at position 62, 64, and five water molecules in the active site. The map is depicted as a blue mesh and contoured at 3 σ for each site-specific mutant. This figure was generated and rendered with PyMOL (www.pymol.org).

Figure 3.

Structural superposition of the active site of the site-specific mutants (A) N62A, (B) N62V, (C) N62T, and (D) N62D with wild-type HCA II. Each panel has the wild-type HCA II (grey) structurally aligned with the respective mutant. Residues are labeled and shown as stick models, the zinc atom is depicted as a white sphere and the oxygens of solvent water molecules as red spheres. The structure of wild-type HCA II used was from PDB accession number 2CBA (11). This figure was generated and rendered with PyMOL (www.pymol.org).

The side chain of the proton shuttle His64 is of particular interest and its dual orientation in HCA II is well documented (9, 10). The inward and outward conformers of His64 in the wild-type structure of Hakansson et al (10) were refined to an occupancy of approximately 70% and 20% respectively, whereas these conformers of His64 in N62T were refined with equal occupancy of 50% each. Full occupancy was assigned to His64 in the case of the mutants that report only a single conformer of His64 (N62A,V,L), although small populations (≤ 10 %) of His64 may be undetected and may assume an alternative conformation. Appropriate B factors are given in Table 2.

Table 2.

Mean B-factors (Ų) for the ordered side chains of His64 and amino acids in its immediate vicinity in the active-site cavity of HCA II and variants.

	Wild typea	N62A	N62V	N62T	N62D
His64	13.2 b/ 8.5 c	24.9 b	19.6 b	12.5 b / 14.7 c	18.2 c
Trp5	11.3	20.4	16.8	14.3	17.2
Try7	8.9	14.9	13.0	9.5	12.1

^aData from the Protein Data Bank, accession code 2CBA solved to a comparable resolution of 1.5 Å.

^b‘In’ conformer.

^c‘Out’ conformer.

In wild type HCA II the inward orientation has dihedral angles χ₁ and χ₂ near 44° and 95°, respectively, while the outward orientation has angles of -39° and 98°. The dihedral angle χ₁ describes the inward and outward orientations of the side chain of His64 representing rotation about the Cα-Cβ bond. For N62A and N62V HCA II, His64 was observed only in the inward conformation, and for N62D only in the outward conformation (Supporting Information, Table SI1). In contrast, the mutant N62T HCA II resembled wild type enzyme by displaying two superimposed conformations for the His64 side chain, the inward and outward. These two side-chain conformations were equally present in the crystal structure of N62T HCA II.

None of the mutations significantly disrupted the active site solvent network compared with wild-type HCA II, although a slight shift of 0.7 Å in the position of the water molecule W3b was observed in all the mutant forms (Figures 2,3). In the case of the hydrophobic substitutions of Ala and Val at position 62, water molecule W3b moved away from the side chain of residue 62 (Figures 3A and B). Hydrophilic substitutions of Asp and Thr caused W3b to move towards the polar side chain of residue 62 (Figures 3C and D). Besides these small positional perturbations, the network of ordered solvent remained conserved (Figure 3). Of all the mutant structures, N62A has slightly higher B factors for the ordered water structure, but this is most likely due to its slightly lower resolution than the others (Supporting Information, Table SI2).

The replacement of Asn62 with Asp and Ala induced shifts in the side chains of nearby residues Asn67 and Gln92 (Supporting Information, Table SI1). The side chain atoms, Oδ1 and Nδ2 of Asn67 in N62D were shifted by 1.4 Å and 0.3 Å respectively towards His64 (Δχ₁ ≈ 25°; Δχ₂ ≈ 37°). In the case of N62A, the same atoms were shifted by 0.8 Å and 0.2 Å respectively towards His64 (Δχ₁ ≈ 25°; Δχ₂ ≈ 31°). The side chain atoms Nη2 of Gln92 in N62D and N62A were shifted by 1.3 Å (Δχ₃ ≈ 33°) and 0.8 Å (Δχ₃ ≈ 20°) respectively.

Catalysis

The catalysis by carbonic anhydrase of ¹⁸O exchange between CO₂ and water yields two rates, as described in Methods. The first is R₁ of eq 5 from which we determined k_cat^ex/K_eff^CO2 for the hydration direction. The pH profile of this rate constant for wild type and mutants at residue 62 were adequately fit to the titration of a single residue (Figure 4) with resulting values of the kinetic pK_a, an estimate of the pK_a of the zinc-bound water (3, 29), given in Table 3. These data show a maximum at high pH corresponding to the reactivity of the zinc-bound hydroxide form of the enzyme in the hydration direction, with the maximal values of k_cat^ex/K_eff^CO2 given in Table 4. The pK_a values were all close to pK_a 7.0 as found in wild type, with the exception of N62D HCA II for which the pK_a was elevated to 7.9 (Table 3, column 5). However, the maximal, pH independent values of k_cat^ex/K_eff^CO2 for the four mutants showed considerable variation with each mutant less than k_cat^ex/K_eff^CO2 for wild type; the value for N62D was the smallest (Table 4). A somewhat more precise fit of the data was achieved by introduction of a second ionization; however, this had minor effects on the resulting parameters of Tables 3 and 4. The values of K_eff^CO2 are large in these experiments, exceeding the solubility of CO₂ (about 34 mM) under our conditions. Hence, k_cat^ex was not able to be determined.

Figure 4.

The pH profiles for k_cat^ex/K_eff^CO2 for the hydration of CO₂ catalyzed by the following variants of HCA II: wild type (●); N62V (○); N62A (■); N62T (▲); and N62D (◆). Data were obtained by ¹⁸O exchange between CO₂ and water measured at 25 °C in solutions containing 25 mM of all species of CO₂ and at sufficient Na₂SO₄ to maintain 0.2 M ionic strength. No buffers were added.

Table 3.

Apparent values of pK_a obtained by various kinetic measurements of catalysis by HCA II and mutants.

Enzyme	Orientation of His64	pKa His64a (eq 6)	pKa ZnH2Oa (eq 6)	pKa ZnH2Ob (eq 5)	pKa ZnH2O (esterase)
wild-type	in/out	7.2	6.8	6.9	6.9
N62T	in/out	7.0	7.0	7.0	7.0
N62D	out	8.4	7.6	7.9	7.7
N62A	in	6.2	6.5	7.2	7.1
N62V	in	5.9	5.9	7.3	7.1
N62Lc	in	6.0 d	6.0 d	7.3	7.1

^aMeasured from the fits of eq 6 to data of Figure 5. The values of pK_a have standard errors generally near ± 0.1 and no greater than ± 0.2.

^bMeasured from the data of Figure 4 using eq 5. The standard errors in pK_a are mostly ± 0.1 and no greater than ± 0.2.

^cThese data from Fisher et al. (18)

^dThese values estimated from poorly resolved pH dependence shown in Fisher et al. (18)

Table 4.

Maximal values of rate constants for hydration of CO₂, for hydrolysis of p-nitrophenylacetate, and for proton transfer in the dehydration direction catalyzed by HCA II and variants.^a

Variant of HCAII	Orientation of His64	(kcatex/KeffCO2)b hydration	kcat/Km esterase	kBb dehyd
		μM-1s-1	M-1s-1	μs-1
wild type	in/out	120	2800	0.80
N62T	in/out	69	2880	0.39
N62D	out	53	1900	0.043
N62A	in	81	2220	0.39
N62V	in	83	2360	0.35
N62Lc	in	140	2050	0.20d

^aThe standard errors for these rate constants are 20% or less.

^bMeasured from the exchange of ¹⁸O between CO₂ and water using eqs 5 and 6.

^cThese data from Fisher et al. (18)

^dThis value estimated from poorly resolved pH dependence shown in Fisher et al. (18)

The values of pK_a determined from k_cat^ex/K_eff^CO2 in the ¹⁸O exchange experiment are very similar to the values of pK_a determined from the pH dependence of k_cat/K_m for the catalyzed hydrolysis of 4-nitrophenylacetate (Table 3, column 6). The variation in the maximal values of k_cat /K_m for ester hydrolysis was less that for k_cat^ex/K_eff^CO2; however, k_cat /K_m for N62D HCA II was the smallest in both sets of data (Table 4). Again, K_m for this ester hydrolysis is too large to determine k_cat.

The values of R_H2O/[E] provide an independent set of parameters that represent a rate constant for the proton transfer dependent release of H₂¹⁸O from the active site (eq 4). R_H2O/[E], the rate of release of H₂¹⁸O from the active site, is determined by intramolecular proton transfer as verified by pH profiles, kinetic isotope effects, and chemical rescue experiments (28, 31). The pH profiles of R_H2O/[E] appear bell shaped for much of the pH range of these studies (Figure 5), a feature that has been interpreted in terms of eq 6 and the transfer of a proton from one prominent donor His64 to the zinc-bound hydroxide (28, 31). The solid lines for the bell-shaped regions of Figure 5 represent fits of eq 6 to the data with the resulting parameters appearing in Tables 3 and 4. In order to apply eq 6 correctly, the assignment of the values of pK_a for the donor and acceptor needs to be established. This was achieved using the values of pK_a for the zinc-bound water pK_{a ZnH2O} as determined by k_cat^ex/K_eff^CO2 from the ¹⁸O-exchange experiment and by k_cat/K_m for the ester hydrolysis, which are in agreement (Table 3, columns 5,6). The ¹⁸O-exchange experiments were carried out at chemical equilibrium in the absence of added buffer. Buffers can participate in the proton transfer and complicate interpretation.

Figure 5.

The pH profiles of R_H2O/[E], the rate constant for the release of H₂¹⁸O from the enzyme, catalyzed by these variants of HCA II: wild type (●); N62V (○); N62A (■); N62T (▲); and N62D (◆). Conditions were as described for Figure 4.

The values of pK_{a ZnH2O} and pK_{a His64} for wild-type and N62T HCA II were nearly identical (Table 3); however, the value of the rate constant for proton transfer k_B for N62T was half that of wild type (Table 4). For N62D HCAII the value of pK_{a ZnH2O} is elevated to 7.6 -7.9 (Table 3) with the value of pK_{a His64} at 8.4 also elevated compared with wild type. This corresponds to His64 predominantly in the outward orientation with k_B very low at 0.04 μs^-1, about 5% of the value of wild type (Table 4).

The remaining variants of Tables 3 and 4, specifically N62A,V, and L, are not as straightforward because the values of pK_{a ZnH2O} near 7.2 determined from k_cat^ex/K_eff^CO2 of the ¹⁸O exchange experiment and k_cat/K_m from the ester hydrolysis (Table 3, columns 5,6) do not agree with the values of pK_aZnH2O determined from R_H2O/[E] which are near 6.0 (Table 3, column 4). The bell-shaped curves for these variants are clearly shifted to lower pH (Figure 5) consistent with values of pK_{a ZnH2O} and pK_{a His64} both near 6.0 (Table 3). The resulting values of k_B are in the range of 0.2 to 0.4 μs^-1, not very different than the corresponding values of the other enzymes of Table 4 (except N62D). The data for N62L HCA II are from a previous report (18) and were not interpreted there because of their complicated pH profile for R_H2O/[E]. However, the data of Figure 5 show pH profiles for N62A and N62V that are less complicated but show features shared by N62L. With this background eq 6 was applied to all three of these variants N62A,V, and L. We note that each of the mutants N62A and N62V (Figure 5), as well as N62L (18), have a secondary, very small maximum for R_H2O/[E] at pH near 8 that resembles the data for N62D. This presumably indicates a second, less efficient ¹⁸O exchange pathway.

DISCUSSION

The goal of this work was to elucidate the role in catalysis of residue 62 on the hydrophilic side of the active-site cavity in HCA II, in particular its influence on the structure of ordered solvent, on the properties of the proton shuttle His64, and on the kinetics of catalysis. An advantage with the amino-acid substitutions we made at position 62 was that each of the resulting mutants had an ordered solvent structure in the active-site cavity very similar to that of wild-type HCA II. This appears to remove solvent structure as a variable in comparing catalysis by these mutants. As a result, these mutants allow this study to focus more completely on the properties of the side chain of His64.

The crystal structures of the variants at residue 62 are closely superimposable with wild-type HCA II (Figure 3). A major difference is in the orientation of the side chain of the proton shuttle residue His64 which appears for certain mutants in an entirely inward or entirely outward conformation, within experimental error (Figure 2, Supporting Information Table SI1). In wild-type HCA II these conformers interchange with a rotation about χ₁ of about 100°. In any case, the kinetic barrier between inward and outward orientations is near 6 kcal/mol in wild-type HCA II according to computations, and is not expected to contribute to rate of catalysis (with an energy barrier near 10 kcal/mol)(32).

For the mutation N62D HCA II, His64 was observed entirely in the outward conformation, within experimental uncertainty (Figures 2, 3). In this case the pK_a for the imidazole of the side chain of His64, determined from pH profiles of catalysis, was elevated to 8.4 compared with wild type which is near 7.2 (Table 3)(33). The elevated pK_a of His64 in the N62D mutants is likely due to the nearby Asp62, possibly in an undetected inward orientation. This is also consistent with the pH dependence of rotamer populations in wild-type HCA II showing the outward orientation favored at lower pH (9, 10). In N62D HCA II, the pK_a of the zinc-bound water is also elevated to 7.6 - 7.9 (Table 3), which is also anticipated based on the shift to the outward orientation of the partially positively charged side chain of His64.

The ordered water structure appears unchanged in N62D compared with wild type although His64 is in the outward orientation in this mutant (Figure 2). It is significant that ΔpK_a between proton donor and acceptor (pK_{a ZnH2O} — pK_{a His64}) is small, near 0.4 – 0.8, for both wild type and N62D HCA II, yet the rate constant for proton transfer k_B for N62D is about 20-fold less than wild type (Table 4). This shows that the lower rate constant k_B for N62D is not due primarily to changes in pK_a, but rather to the much lower capacity for proton transfer of His64, presumably because of its greater distance from the zinc (about 12 Å). This is consistent with accumulated evidence from other studies of the distance dependence of the proton transfer in carbonic anhydrase (34). The longer distance between His64 and the zinc-bound hydroxide in N62D (∼ 12 Å) is suggested to have a slower proton transfer at least in part because of the energy inherent in constructing longer water wires for proton transfer (32). In addition, computations show that the outward orientation of His64 is associated with shorter lifetimes of the ordered water molecules in the active-site cavity (19). Hence, the lower rate constant for proton transfer k_B for N62D can be attributed to His64 in the outward orientation. However, data on this topic are complex. Some computations indicate that proton transfer in carbonic anhydrase is dominated by electrostatics and is not expected to be dependent on distance (15, 35). Introducing a charged group such as Asp62 near the proton transfer pathway would be expected to alter the electrostatic properties of the donor and acceptor, as noted above, but also the apparent pK_a values of water molecules in the proton transfer pathway. This could contribute to the low efficiency of proton transfer in catalysis by N62D. The orientation of His64 in T200S HCA II is outward yet the steady-state constants for catalysis are very similar to those of wild-type HCA II (36).

For each of the three mutants in which His64 is in the inward orientation (N62A, N62V, N62L) the pK_a for His64 determined from R_H2O is decreased to near 6.0 (Table 3), due in part to the position of the imidazole ring in a more hydrophobic environment and more sequestered from bulk solvent (Figure 2). The pK_a for the zinc-bound water determined in these mutants from R_H2O/[E] is considerably smaller than in wild type (Table 3). This decrease is expected in these three mutants because of greater stabilization of the zinc-bound hydroxide by the inward oriented imidazolium form of His64. However, in contrast to the smaller value of pK_a near 6 determined from R_H2O/[E], the pK_a values for zinc-bound water observed from k_cat^ex/K_eff^CO2 and of k_cat/K_m of the esterase catalysis are near 7 (Table 3). These data imply that in the transition state for proton transfer (in N62A, N62V, N62L), the pK_a values of the proton acceptor is low compared with the pK_a determined from the first stage of catalysis (eq 3). In each case however, the rate constant for proton transfer appears close to the same (0.20 to 0.39 μs^-1, Table 4). This probably indicates that it is the inward orientation that is responsible for proton transfer between His64 and the zinc-bound solvent molecule in catalysis.

Finally, the values of pK_a for donor and acceptor for N62T HCA II appear very close to those for wild type (Table 3). Both of these enzymes show appreciable occupancy of His64 in the inward and outward orientations. Perhaps it is significant that these pK_a values are midway between those found for His64 in the mutants for which this residue lies entirely in the inward and entirely in the outward orientations. However, the rate constant for proton transfer k_B is less by about two-fold for N62T compared with wild type (Table 4).

An interesting feature of the data of Figure 4 compared with an earlier study (18), is that in this study replacement of Asn62 with hydrophobic residues caused substantial changes in k_cat^ex/K_eff^CO2 (Table 4). In the case of N62D, a decrease as much as two-fold in the maximal values of k_cat^ex/K_eff^CO2 for hydration was observed (Table 4). This is not explained for N62A,V, and L by values of the pK_a of the zinc-bound water which are arguably the same as in wild type. Moreover, the decrease in k_cat^ex/K_eff^CO2 for N62D is associated with a more basic pK_a for the zinc-bound water than in wild type (Table 3). Since these mutations alter the charge density in the active-site cavity, electrostatic effects on the transition state for the nucleophilic attack on CO₂ or on the dissociation of bicarbonate could be responsible.

Conclusions

Replacment of Asn62 with a number of amino-acids maintained the ordered water structure observed by x-ray crystallography in the active-site cavity of HCA II. This removed water structure as a variable in comparing with wild type the activities of mutants at residue 62. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that are necessary for maximal efficiency of this enzyme in transferring protons between the active site and solution (34, 37). The site-specific mutant N62D was observed to have an outward orientation of His64, yet the difference in pK_a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II in the dehydration direction was 5% that of wild type, showing that His64 in this mutant is inefficient in proton transfer compared with wild type because of its predominantly outward orientation compared with wild type which shows both inward and outward orientations. Additional contributions to inefficient proton transfer could arise from nonspecific electrostatic effects due to introduction of a charged group Asp62 near the proton transfer pathway. These results emphasize the role of Asn62 among the residues on the hydrophilic side of the active-site cavity in maintaining efficient catalysis by carbonic anhydrase.

Abbreviations

HCA II

isozyme II of human carbonic anhydrase

N62A

a site-specific mutant in which Asn62 is replaced by Ala

RMSD

root mean square deviation