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* Department of Pharmacology and Therapeutics, Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida
Correspondence: Address reprint requests to D. N. Silverman, E-mail: silvermn{at}college.med.ufl.edu; or R. McKenna, E-mail: rmckenna{at}mbi.ufl.edu.
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ABSTRACT |
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8 Å from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp-64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of approximately fourfold greater activation with the mutant containing Trp-64 compared with Gly-64, and the observation by x-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp-64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp-64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp-64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions wr and wp rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis. |
INTRODUCTION |
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-class carbonic anhydrases in the hydration of CO2 and dehydration of bicarbonate occurs in two stages shown in Eqs. 1 and 2 (1
–4). The first stage is the conversion of CO2 into bicarbonate by reaction with a zinc-bound hydroxide; the dissociation of bicarbonate leaves a water molecule at the zinc (Eq. 1).
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 | (1) |
There is no evidence of rate-contributing proton transfer in the steps of Eq. 1 (5,6). The second stage is the transfer of a proton to solution to regenerate the zinc-bound hydroxide (Eq. 2); here B denotes an exogenous proton acceptor from solution or a residue of the enzyme itself such as His-64 that subsequently transfers the proton to solution (7,8).
 | (2) |
Crystal structures of HCA II have shown that the side chain of His-64 can occupy two conformations, an inward orientation in which the imidazole side chain is 7 Å away from the zinc and an outward orientation at 12 Å away from the zinc. There appears to be little barrier to interconversion of these rotamers, and it has been suggested that this flexibility contributes to efficient proton transfer (9,10). The distance between the imidazole side chain of His-64 in either conformation and the zinc-bound solvent molecule is too distant for direct proton transfer (2,9,10), and it appears that proton transfer must proceed through intervening water molecules (11).
Binding sites in the active-site cavity HCA II and mutants have been found for small molecules suggested to be activators of catalysis (12–16). Two binding sites for the activator 4-methylimidazole (4-MI) were found, one associated with the side chain of Trp-5 (12) and a second with the side chains of Glu-69 and Asp-72 (16). The issue of whether the sites determined by crystallography were productive binding sites was investigated by altering these binding sites through mutagenesis and determining whether small molecule rescue was changed. It was determined that the binding sites of 4-MI associated with the side chains of Trp-5 (12) and Asp-72 (unpublished) did not contribute to proton transfer in catalysis. A reasonable explanation is that they were too far (>12 Å) from the zinc for productive proton transfer. These studies suggested whether a mutant of HCA II could be engineered with a binding site for small molecule rescue placed closer to the zinc, since, at residue 64 in a mutant containing H64W, the energetic features of the activation could be examined by variation of the rescuing proton donor.
In this report, we describe the structural details of a binding site at Trp-64 in small molecule rescue of a mutant of HCA II, a binding site that is demonstrated to be a productive participant in catalysis by comparing activation of H64W HCA II with that of H64G HCA II. The binding of 4-MI appears in a 
-stacking interaction with the indole side chain of Trp-64 as determined from an x-ray crystal structure. The activation of H64W and H64G HCA II by derivatives of imidazole and pyridine are presented in free energy plots, which are interpreted by analogy with nonenzymic, bimolecular proton transfer, and in terms of Marcus rate theory (17–19). The results emphasize the role of solvation in the enhancement of catalysis caused by small molecule rescue. The data identify regions of the active-site cavity from which proton transfer is efficient and contribute to our understanding of catalysis influenced in rate by solvent rearrangement and a facile proton transfer step with low kinetic barrier.
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METHODS |
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). The sequences were confirmed from the DNA of the entire coding region for carbonic anhydrase in the expression vector. Carbonic anhydrases were purified using affinity chromatography with p-(aminomethyl)-benzene-sulfonamide coupled to agarose beads (21), followed by extensive dialysis. Matrix-assisted, laser desorption ionization time-of-flight mass spectrometry gave results consistent with the protein sequences. Purity of enzyme samples was determined by electrophoresis on a 10% polyacrylamide gel stained with Coomassie blue. Concentrations of HCA II and mutants were determined from the molar absorptivity at 280 nm (5.5 x 104 M–1 cm–1), and by titration with the tight binding inhibitor ethoxzolamide while observing enzyme-catalyzed 18O exchange between CO2 and water. These measurements gave close agreement.
Crystallography
Crystals of mutants of HCA II were obtained using the hanging drop vapor-diffusion method (22). The crystallization drops were prepared by mixing 5 µL of protein (concentration 20.0 mg/mL in 50 mM Tris-HCl, pH 7.8) with 5 µL of the precipitant solution (50 mM Tris-HCl, pH 8.2, and 2.3 M ammonium sulfate) at 277 K and allowed to equilibrate against 1000 µL of the precipitant solution. For the co-crystals containing 4-MI and W5A-H64W HCA II, the crystallization buffer was prepared as described above with the addition of 50 mM 4-MI. Useful crystals were observed five days after the crystallization setup. X-ray diffraction data sets were collected using an R-AXIS IV++ image plate system with Osmic mirrors and a Rigaku HU-H3R Cu rotating anode operating at 50 kV and 100 mA (Rigaku, The Woodlands, TX). The crystals were quick-dipped in a cryoprotectant (30% glycerol in precipitant solution) before flash-freezing using an Oxford Cryosystems device (Devens, MA), and all data sets were collected at 100 K. The crystal-to-detector distance was set to 80 mm for H64W and 100 mm for W5A-H64W HCA II with and without 4-MI. The oscillation steps were 1° with a 10-min exposure per image. X-ray data indexing was performed using DENZO and scaled and reduced with SCALEPACK (23). Data collection statistics for each resolution bin are given in Supplementary Material Table S1. All models were built using O, ver. 7 (24). Refinement was carried out with CNS, ver. 1.1 (25). The isomorphous wild-type HCA II crystal structure (Protein Data Bank entry 2CBA (26) was used to phase the data sets. To avoid phase-bias of the model, the zinc ion, mutated side chains, 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, the 2Fo-Fc Fourier electron density maps were generated. These electron density maps clearly showed the position of the zinc ion and the mutated residues, which were subsequently built into their respective density. For the W5A-H64W HCA II and 4-MI cocrystal data, Fo-Fc Fourier electron density maps were generated and the 4-MI molecule was placed in the electron density and subsequently included in refinement cycles. After several cycles of temperature factor and energy minimization refinement, solvent molecules were incorporated into the models using the automatic water-picking program in CNS until no more water molecules were found at a 2.0 
contour level. Refinement of the models continued until convergence of Rcryst and Rfree was reached. Refinement and model statistics are given in Table 1.
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and water at chemical equilibrium (27,28) (Eqs. 3 and 4). The 18O-exchange method has the advantage that it is carried out at chemical equilibrium and pH control is not a problem; very low as well as very large concentrations of proton donors/acceptors can be used (27).
 | (3) |
 | (4) |
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) was used to measure the isotopic content of CO2. Solutions contained 25 mM total concentration of all species of CO2 unless otherwise indicated. All experiments were performed at 25°C and in all experiments ionic strength was held at a minimum of 0.2 M by addition of sodium sulfate.
This approach yields two rates for the 18O exchange catalyzed by carbonic anhydrase (27). The first is R1, the rate of exchange of CO2 and 
at chemical equilibrium (Eq. 3), as shown in Eq. 5.
 | (5) |
Here 
is a rate constant for maximal interconversion of substrate and product, 
is an apparent binding constant for substrate to enzyme, and [S] is the concentration of substrate, either CO2 or bicarbonate (27). The ratio 
is, in theory and in practice, equal to kcat/Km obtained by steady-state methods.
A second rate determined by the 18O exchange method is RH2O, the rate of release from the enzyme of water-bearing substrate oxygen (Eq. 4). This is the component of the 18O exchange that is enhanced by exogenous proton donors (8,27). In such enhancements, the exogenous donor acts as a second substrate in the catalysis providing a proton (Eq. 4), and the resulting effect on 18O exchange is described by Eq. 6 below.
 | (6) |
Here 
is the observed maximal rate constant for the release of 
to bulk water caused by the addition of the buffer. 
is an apparent binding constant of the buffer to the enzyme, [E] and [B] are the concentrations of total enzyme and total buffer, and 
is the rate of release of into solvent water in the absence of added buffer and represents the contribution to proton transfer from other sites on the enzyme or possibly solvent water itself.
With the addition of derivatives of imidazole and pyridine at concentrations up to 200 mM, we have observed a weak inhibition of both R1 and RH2O. This is probably due to binding at or in the vicinity of the zinc in the manner found for the binding of imidazole to carbonic anhydrase I (29) and 4-MI to a mutant of HCA II (30). The binding constant Ki for this inhibition is generally >100 mM, indicating weak binding at the inhibitory site. Some exogenous donors (e.g., 1,2-dimethylimidazolium; 3,4-dimethylpyridinium) exhibited no inhibition. In each case of inhibition, a single value Ki described inhibition of both R1 and RH2O, as determined by these equations:
 | (7) |
The pH dependence of RH2O/[E] is often bell-shaped, consistent with the transfer of a proton from a single predominant donor to the zinc-bound hydroxide. In these cases, the pH profile is adequately fit by Eq. 8 in which kB is a pH-independent rate constant for proton transfer, and (Ka)donor and (Ka)ZnH2O are the noninteracting ionization constants of the proton donor BH of Eq. 4 and the zinc-bound water.
 | (8) |
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for hydration of CO2 (Eqs. 3 and 5) and the rate constant RH2O/[E] for the proton transfer-dependent release of from the enzyme during catalysis (Eqs. 4 and 6) (27).
The values of for hydration of CO2 catalyzed by H64G, H64W, and W5A-H64W HCA II were not activated by increasing concentrations (up to 200 mM) of the derivatives of imidazole and pyridine used (typical data shown in Fig. 1). This is consistent with the catalytic mechanism in which the conversion of CO2 into bicarbonate does not contain rate-contributing proton transfers. The maximal values of at 108 M–1 s–1 for catalysis by H64W HCA II (Fig. 1) were as great as that observed for wild-type enzyme (5). We sometimes observed a weak inhibition in caused by addition of imidazole derivatives with values of the inhibition constant Ki between 150 mM and 250 mM. This effect has been observed by Elder et al. (30) for the binding of 4-MI to H64A HCA II as a second sphere ligand of the zinc. In such cases, the inhibition was taken into account in estimating , as described in Methods (Eq. 7).
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). The plot in Fig. 3 representing the difference between RH2O/[E] in the presence and absence of 4-MI can be fit by Eq. 8, which describes activation as a single proton transfer between noninteracting donor and acceptor groups. This difference plot represents the activation of H64W HCA II by 4-MI and is fit by a value of 
with pKa values of 6.7 ± 0.1 and 8.3 ± 0.1 (Fig. 3). These are to be compared with the pKa of 7.3 ± 0.1 measured for the zinc-bound water determined from the pH profile of catalyzed by H64W HCA II (data not shown), and with the solution pKa of 7.9 for 4-MI. The data of Figs. 2 and 3 confirm that the activation observed for 4-MI is consistent with proton transfer. The rate constants 
and kB were determined by fitting (Enzfitter, Biosoft, Cambridge, UK) of Eqs. 6–8 to data for each activator such as shown in Figs. 2 and 3.
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pKa in Fig. 4 to comment on the existence of an inverted region.
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atoms. Final model refinement statistics are given in Table 1. Attempts to observe binding of imidazole, 4-methylimidazole, and 3,5-dimethylpyridine to H64W HCA II, and imidazole and 3,5-dimethylpyridine to W5A-H64W HCA II, were unsuccessful. These attempts were done by cocrystallization and soaking crystals in solutions containing these proton donors. Coordinates and structure factors for H64W, W5A-H64W, and W5A-H64W with bound 4-MI have been deposited with the Protein Data Bank (PDB; http://www.rcsb.org) with the accession codes 2FNK, 2FNM, and 2FNN, respectively.
Active-site architecture of HCA II mutants
The single mutant, H64W HCA II, was structurally similar to wild-type, with the exception of active site residues Trp-5 and Trp-64. These two residues are in close proximity to each other and appear to loosely -stack with each other in H64W HCA II (Fig. 5 A). The side chains of Trp-5 and Trp-64 were somewhat disordered as reflected in the observed B-factors (25 and 30 Å2, respectively) compared to all side chains (16 Å2) and by visual inspection of the electron density (Table 1, Fig. 5 A). This apparent disorder could be due to steric hindrance between the residues or dynamic movement of these two bulky hydrophobic side chains. Replacement of Trp-5 with Ala led to significantly improved order in the electron density for the side chain Trp-64 in the W5A-H64W crystal structure, making it possible to place the side chain unequivocally (Fig. 5 B).
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3.5 Å from Trp-64 was observed. The 4-MI -stacked with the side chain of Trp-64 and made a hydrogen bond between the N3 atom and O
1 of Thr-200 (Fig. 5, C and D). The 4-MI is 8 Å away from the zinc ion in the active site (Fig. 5 C). It is noteworthy that in both H64W and W5A-H64W HCA II, the side chain of Trp-64 seems to occupy a similar conformation to the inward position of His-64 in wild-type HCA II (Fig. 6) (9,10). The 4-MI molecule bound in W5A-H64W HCA II adopts a position that seems to straddle the inward and outward positions of His-64 in wild-type HCA II (Fig. 6) (9,10). This shows 4-MI bound at Trp-64 closer by 4 Å than when bound at Trp-5 in H64A HCA II (Fig. 6) (13).
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) consisting of the Zn-OH–/H2O, W1, W2, W3a, and W3b, was conserved in the mutants of HCA II in Fig. 5, A–C. In H64W HCA II and in W5A-H64W HCA II without 4-MI, solvent molecule W3a is within hydrogen-bonding distance (2.9 and 2.8 Å, respectively) of the backbone O of Trp-64 (Fig. 5, A and B). This is in contrast with wild-type structures where the distances between W2, W3a, and W3b are all too long (3.5 Å) to constitute viable hydrogen bonds with His-64 (10). |
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12 Å from the zinc (12,13); 2), in a pocket formed by the side chains of Glu-69, Ile-91, and Asp-72 13 Å from the zinc (16); and 3), as a second-shell ligand of the metal in CO(II)-substituted H64A HCA II, a site determined to be inhibitory to catalysis (30).
Since these binding sites for 4-MI were nonproductive in proton transfer, we used another strategy and constructed a binding site for small molecule rescue near the site of His-64, the proton shuttle of wild-type enzyme. We have examined a variant of HCA II with His-64 replaced by Trp. Previous studies had emphasized replacement of His-64 with Ala (12,13); this replacement with Trp is unique. The crystal structure of H64W HCA II is superimposable with wild-type and shows the extension into the active-site cavity of the indole ring of Trp-64 (Fig. 5 A). The strategy is based on the expectation that the proton donor 4-MI will bind to Trp-64 8 Å from the zinc and will contribute to proton transfer (to an extent larger than for H64G) being at a location that is near the proton shuttle residue His-64 in the inward conformation (9,10). Trp-64 itself is not a proton transfer residue.
Small molecule rescue of catalysis
The expected activation has been observed. A series of experiments show the enhancement of catalysis by H64W HCA II upon addition of exogenous proton donors. These experiments include saturable activation of the second stage of catalysis (Eq. 4; Fig. 2), solvent H/D isotope effects on maximal activation near 2 (Fig. 2), pH profiles consistent with proton transfer between exogenous donors/acceptors and the zinc-bound aqueous ligand (Fig. 3), observation of a much smaller activation with H64G than H64W HCA II (Fig. 2), and finally the observation by x-ray crystallography of the binding of 4-MI associated in a -stacked interaction with the indole side chain of Trp-64 in W5A-H64W HCA II (Figs. 5 and 6). This is reasonable evidence that activation by 4-MI, and most likely the other activators of Fig. 4, occurs in part by binding to Trp-64 with subsequent participation in proton transfer.
It is interesting that x-ray crystallography did not show the binding of exogenous donors to H64W HCA II, the mutant that was activated by these donors. However, the binding of 4-MI to W5A-H64W HCA II was observed even though this mutant was activated to a lesser extent (Fig. 2). The distance between the indole rings of Trp-5 and Trp-64 in H64W CA II is 3.5 Å, too close for 4-MI to bind between them without steric clashes. However, this distance between the two rings is essentially what is observed in the base-stacking of B-DNA. Hence it is conceivable that 4-MI could bind like a DNA intercalator to open the gap between Trp-5 and Trp-64 and sandwich between them. However, we did not observe such a structure in crystals of H64W in the presence of 4-MI and it is possible that the protein backbone structure is too rigid to allow such a structural rearrangement to occur.
The lack of an observed binding site for 4-MI in the crystal structure of H64W HCA II might be related to the conformational flexibility and disorder of the side chain of Trp-64 in this mutant. The electron density corresponding to the side chain of Trp-64 in H64W HCA II is smeared and has a mean thermal B-factor near 30 Å2, which is twice as large as the other side chains in the active site and therefore can be interpreted to reflect conformational mobility of this side chain (Fig. 5 A). This mobility may be necessary to achieve a conformation from which proton transfer can proceed, whereas a more constrained conformation that is not favorable for proton transfer would be associated with lower catalytic activity. Interestingly, Trp-64 in the double mutant W5A-H64W HCA II without and with 4-MI bound is more ordered with mean thermal B-factors near 16 and 14 Å2, respectively (Fig. 5, B and C; Table 1). This mutant is weakly activated compared with H64W HCA II (Fig. 2). Conformational mobility appears in several studies to be associated with efficient proton transfer in catalysis by carbonic anhydrases (9,10,32,33), and these data are yet another example.
This study locates a binding site for small molecule rescue of carbon anhydrase, the first case that is demonstrated by the decrease of activation when the binding site is altered through mutagenesis. Also, this site is located in the vicinity of His-64 of the wild-type enzyme (Fig. 6), not at more distant or closer sites, suggesting a significant distance-dependence. Table 3 places these results in the context of previous studies and provides a rough quantitation of the distance requirements for small molecule rescue of proton transfer in this system.
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). In this respect, the properties of the nonenzymic proton transfers are similar to those accompanying catalysis by carbonic anhydrase. The most straightforward quantitation of the barriers is through application of classical Marcus theory applied to proton transfer, which has been explored in the case of nonenzymic transfers (18).
In Marcus theory applied to proton transfer, the observed overall activation energy for proton transfer G
is described in terms of the standard free energy of reaction G° and an intrinsic kinetic barrier 
, which is the value of G when G° = 0, that is, when the transfer is free of thermodynamic influences and represents a pure or "intrinsic" energy barrier (17,18). The Marcus equation is further modified to describe proton transfers in which there is a component of the observed activation barrier that does not depend on G° for the reaction. This component is called the work term "wr" for the forward direction (dehydration here). Similarly, "wp" is the work term required for the reverse reaction. In nonenzymic, bimolecular proton transfers, the work term is considered part of the free energy of reaction needed to bring the reactants together and form the reaction complex with associated solvation changes before proton transfer (19). The Marcus equation then becomes
 | (9) |
This expression assumes that the work terms wr and wp as well as the intrinsic energy barrier do not vary for proton transfer between the series of homologous proton donors to which the equation is fit. Catalysis by variants of HCA III (34) and HCA II (12) has previously been interpreted in terms of the Marcus formalism.
The solid lines of Fig. 4 are fits of this Marcus theory describing activation of catalysis (kB of Eq. 8) by proton transfer from a series of imidazole and pyridine derivatives. The corresponding parameters for the fit to the Marcus equation (Eq. 9) (Enzfitter, Biosoft) are given in Table 2.
For the activation of the variants of carbonic anhydrase in Table 2, the values of the Marcus parameters show rather small values of the intrinsic kinetic barrier and large values of the work terms wr and wp, consistent with previous applications of Marcus theory to catalysis by carbonic anhydrase (12,34). The implications for catalysis of a low intrinsic barrier and large work functions have been discussed previously (19,34). These are viewed as estimates and limited by applicability of the classical Marcus theory, which is the most straightforward although certainly not the only interpretation of free energy plots such as Fig. 4. Braun-Sand et al. (35) have analyzed proton transfer in carbonic anhydrase III using an empirical valence bond approach to a multistate model with larger estimates of the intrinsic barrier.
What is new in this work is the comparison of the parameters of the Marcus equation for the small molecule rescue of H64W and H64G. It is apparent from the data in Table 2 that proton transfer from exogenous donors in H64G HCA II has larger values of the work functions wr and wp compared with those of H64W HCA II; however, the Marcus intrinsic barriers are the same for both. This is evident from observation of the similar curvature of the plots in Fig. 4; the curvature of these plots is a direct measure of the Marcus intrinsic barrier: 
. This suggests that the presumed binding in small-molecule rescue to the side chain of Trp-64, as observed with 4-MI (Figs. 5 and 6), allows for more facile formation of a proton transfer pathway through water structure and positioning of proton donors compared with binding sites in H64G HCA II, and that this is reflected in the work functions wr and wp rather than in the intrinsic Marcus barrier itself. There may be other binding sites for 4-MI within the active-site cavity that can transfer protons to the zinc-bound hydroxide, as indicated by activation of H64G, but the presence of Trp-64 has enhanced the concentration of proton donors in a position for facile proton transfer.
In the case of proton transfer by carbonic anhydrase, wr can reasonably be expected to include the energy to orient the proton donor and to form not only the hydrogen-bonded water bridge but also the water structure in the active-site cavity that interacts with the bridge and with the residues of the cavity (19). The large value of wr found for catalysis by carbonic anhydrase does not necessarily indicate the presence of a high energy intermediate or specific active-site conformation (36). It should be noted that the proton transfer step alternates in the mechanism with the interconversion of CO2 and bicarbonate (Eqs. 1 and 2), and that the diffusion of these reactants/products into and out of the active site would surely disrupt solvent structure.
Although 4-MI binds to the side chain of Trp-64, it does not have the efficiency of His-64 in proton transfer. Fig. 3 shows that the maximal rate constant for proton transfer from 4-MI bound at Trp-64 is 0.13 s–1, to be compared with 0.8 s–1 for His-64 in wild-type HCA II (13). The distance of 7 Å between His-64 and the zinc-bound water is a distance that can be spanned by two or perhaps three water molecules. In fact, this is what is observed with the ordered water molecules in the crystal structures (Fig. 5), and Table 3 gives an indication of the sensitivity of the system to the number of water molecules. These considerations appear to be consistent with recent computational estimates of how the efficiency of proton transfer in a model of carbonic anhydrase is dependent on the number of intervening water molecules (37–39). The ordered water in the crystal structures of carbonic anhydrase may not show the exact path for proton transfer but certainly offers important clues. Not surprisingly, evolution has placed the proton shuttle His-64 at an efficient location. A proton shuttle farther from the metal would be unfavorable in requiring alignment of many hydrogen-bonded water molecules, and closer to the metal would not be an efficient position to transfer protons out to solution, and might be inhibitory if too close to the metal.
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ACKNOWLEDGEMENTS |
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This work is supported by the National Institutes of Health, grant No. GM 25154.
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FOOTNOTES |
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Submitted on July 12, 2006; accepted for publication September 27, 2006.
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REFERENCES |
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for catalysis by (
) H64W HCA II; and (•) H64G HCA II as enhanced by the following exogenous proton donors. Values in parentheses are the solution pKa obtained by titration at 0.2 M ionic strength: (a) 1,2-dimethylimidazole (pKa 8.3), (b) 2-methylimidazole (8.2), (c) 2-ethylimidazole (8.0), (d) 4-methylimidazole (7.9), (e) 1-methylimidazole (7.3), (f) imidazole (7.0), (g) 3,4-dimethylpyridine (6.7), (h) 4-methylpyridine (6.1), and (i) 3-methylpyridine (5.8). These measurements were made a 25°C. The value of the pKa of the zinc-bound water in H64W HCA II is 7.3 and for H64G, 7.2 under these conditions determined from its pH profile in kcat/Km for hydration of CO2. Each measurement was made at a pH equivalent to the pKa of the donor. Ionic strength in each measurement was maintained at a minimum of 0.2 M by addition of sodium sulfate. Solid lines in both plots are fits to the Marcus equation (9
) 4-methylimidazole at pH 7.9; (
) 3,4-dimethylpyridine at pH 6.7; (
) 4-methylpyridine at pH 6.1; and (
by 4-methylimidazole of 18O exchange catalyzed by (
) H64W; (
) W5A-H64W; (•) H64G; and (
and 
. For the data in D2O, a fit of 
and 
.
). Data representing the difference between these two sets are shown (
for the proton donor(s) was 8.3 ± 0.1 and pKa for the zinc-bound water was 6.7 ± 0.1.
