Speeding up proton transfer in a fast enzyme: kinetic and crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase II

Catalysis of the hydration of CO2 by human carbonic anhydrase isozyme II (HCA II) is sustained at a maximal catalytic turnover of 1 mus-1 by proton transfer between a zinc-bound solvent and bulk solution. This mechanism of proton transfer is facilitated via the side chain of His64, which is located 7.5 A from the zinc, and mediated via intervening water molecules in the active-site cavity. Three hydrophilic residues that have previously been shown to contribute to the stabilization of these intervening waters were replaced with hydrophobic residues (Y7F, N62L, and N67L) to determine their effects on proton transfer. The structures of all three mutants were determined by X-ray crystallography, with crystals equilibrated from pH 6.0 to 10.0. A range of changes were observed in the ordered solvent and the conformation of the side chain of His64. Correlating these structural variants with kinetic studies suggests that the very efficient proton transfer (approximately 7 micros-1) observed for Y7F HCA II in the dehydration direction, compared with the wild type and other mutants of this study, is due to a combination of three features. First, in this mutant, the side chain of His64 showed an appreciable inward orientation pointing toward the active-site zinc. Second, in the structure of Y7F HCA II, there is an unbranched chain of hydrogen-bonded waters linking the proton donor His64 and acceptor zinc-bound hydroxide. Finally, the difference in pKa of the donor and acceptor appears favorable for proton transfer. The data suggest roles for residues 7, 62, and 67 in fine-tuning the properties of His64 for optimal proton transfer in catalysis.     

Chemical rescue in catalysis by human carbonic anhydrases II and III

The maximal velocity of catalysis of CO(2) hydration by human carbonic anhydrase II (HCA II) requires proton transfer from zinc-bound water to solution assisted by His 64. The catalytic activity of a site-specific mutant of HCA II in which His 64 is replaced with Ala (H64A HCA II) can be rescued by exogenous proton donors/acceptors, usually derivatives of imidazole and pyridine. X-ray crystallography has identified Trp 5 as a binding site of the rescue agent 4-methylimidazole (4-MI) on H64A HCA II. This binding site overlaps with the "out" position in which His 64 in wild-type HCA II points away from the zinc. Activation by 4-MI as proton donor/acceptor in catalysis was determined in the dehydration direction using (18)O exchange between CO(2) and water and in the hydration direction by stopped-flow spectrophotometry. Replacement of Trp 5 by Ala, Leu, or Phe in H64A HCA II had no significant effect on enhancement by 4-MI of maximal rate constants for proton transfer in catalysis to levels near 10(5) s(-1). This high activity for chemical rescue indicates that the binding site of 4-MI at Trp 5 in H64A HCA II appears to be a nonproductive binding site, although it is possible that a similarly effective pathway for proton transfer exists in the mutants lacking Trp 5. Moreover, the data suggest that the out position of His 64 considered alone is not active in proton transfer in HCA II. In contrast to isozyme II, the replacement of Trp 5 by Ala in HCA III abolished chemical rescue of k(cat) by imidazole but left k(cat)/K(m) for hydration unchanged. This demonstrates that Trp 5 contributes to the predominant productive binding site for imidazole, with a maximal level for the rate constant of proton transfer near 10(4) s(-1). This difference in the susceptibility of CA II and III to chemical rescue may be related to the more sterically constrained and electrostatically positive nature of the active site cavity of CA III compared with CA II. The possibility of nonproductive binding sites for exogenous proton donors offers an explanation for the unusually low value of the intrinsic kinetic barrier obtained by application of Marcus theory to chemical rescue of H64A HCA II.     

Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II

In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of (18)O between CO(2) and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10(5) s(-)(1) was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 10(3) s(-)(1) was observed for His62. The observation, in the crystal structures, of a completed hydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does not appear to be required for efficient proton transfer. The data suggest that the number of intervening water molecules between the donor and acceptor supporting efficient proton transfer in HCA II is important, and furthermore suggest that a water bridge consisting of two intervening water molecules is consistent with efficient proton transfer.     

Proton transfer in a Thr200His mutant of human carbonic anhydrase II

Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc-bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A-T200H HCAII), effectively relocating the proton shuttle residue 2 A closer to the zinc-bound hydroxide compared to wild type HCA II. The crystal structure of H64A-T200H HCA II at 1.8 A resolution shows the side chain of His200 directly hydrogen-bonded with the zinc-bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration-dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A-T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc-bound hydroxide, in analogy with proton transfer involving His64 in wild-type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc-bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen-bonded to the zinc-bound hydroxide, can adopt the correct distance geometry to support proton transfer     

Structural and kinetic analysis of the chemical rescue of the proton transfer function of carbonic anhydrase II

Histidine 64 in human carbonic anhydrase II (HCA II) functions in the catalytic pathway of CO(2) hydration as a shuttle to transfer protons between the zinc-bound water and bulk water. Catalysis of the exchange of (18)O between CO(2) and water, measured by mass spectrometry, is dependent on this proton transfer and was decreased more than 10-fold for H64A HCA II compared with wild-type HCA II. The loss of catalytic activity of H64A HCA II could be rescued by 4-methylimidazole (4-MI), an exogenous proton donor, in a saturable process with a maximum activity of 40% of wild-type HCA II. The crystal structure of the rescued complex at 1.6 A resolution shows 4-MI bound in the active-site cavity of H64A HCA II, through pi stacking interactions with Trp 5 and H-bonding interactions with water molecules. In this location, 4-MI is about 12 A from the zinc and approximates the observed "out" position of His 64 in the structure of the wild-type enzyme. 4-MI appears to compensate for the absence of His 64 and rescues the catalytic activity of the H64A HCA II mutant. This result strongly suggests that the out conformation of His 64 is effective in the transfer of protons between the zinc-bound solvent molecule and solution.     

Structural and kinetic analysis of proton shuttle residues in the active site of human carbonic anhydrase III

We report the X-ray crystal structures and rate constants for proton transfer in site-specific mutants of human carbonic anhydrase III (HCA III) that place a histidine residue in the active-site cavity: K64H, R67H, and K64H-R67N HCA III. Prior evidence from the exchange of 18O between CO2 and water measured by mass spectrometry shows each mutant to have enhanced proton transfer in catalysis compared with wild-type HCA III. However, His64 in K64H and K64H-R67N HCA III have at most a capacity for proton transfer that is only 13% that of His64 in HCA II. This reduced rate in mutants of HCA III is associated with a constrained side-chain conformation of His64, which is oriented outward, away from the active-site zinc in the crystal structures. This conformation appears stabilized by a prominent pi stacking interaction of the imidazole ring of His64 with the indole ring of Trp5 in mutants of HCA III. This single orientation of His64 in K64H HCA III predominates also in a double mutant K64H-R67N HCA III, indicating that the positive charge of Arg67 does not influence the observed conformation of His64 in the crystal structure. Hence, the structures and catalytic activity of these mutants of HCA III containing His64 account only in small part for the lower activity of this isozyme compared with HCA II. His67 in R67H HCA III was also shown to be a proton shuttle residue, having a capacity for proton transfer that was approximately four times that of His64 in K64H HCA III. This is most likely due to its proximity and orientation inward towards the zinc-bound solvent. These results emphasize the significance of side chain orientation and range of available conformational states as characteristics of an efficient proton shuttle in carbonic anhydrase.     

Role of hydrophilic residues in proton transfer during catalysis by human carbonic anhydrase II

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 CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A 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, N62V, N62L) 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 p K 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.     

Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis

Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides. We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme. Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes. Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry. We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III. The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups. Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64. Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.     

A proposal for the mechanism of carbonic anhydrase action

A new catalytic mechanism is proposed for the hydration of CO₂ by the zinc metalloenzyme carbonic anhydrase. This mechanism identifies the group controlling catalytic activity as an active site histidine, in which the protonated imidazole ring coordinates to zinc, losing a proton. Geometric constraints on the histidine unit make the metal-ligand bond a strained and, therefore, labile one. In the hydration reaction, the metal-bound neutral histidine moiety serves as a proton acceptor for the transient ionization of metal-bound water. Zinc-bound hydroxide attacks the carbon of the substrate to generate metal-bound bicarbonate, and the system regenerates itself by losing the elements of carbonic acid.     

Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase

We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.     

Proton transfer from exogenous donors in catalysis by human carbonic anhydrase II

In the site-specific mutant of human carbonic anhydrase in which the proton shuttle His64 is replaced with alanine, H64A HCA II, catalysis can be activated in a saturable manner by the proton donor 4-methylimidazole (4-MI). From 1H NMR relaxivities, we found 4-MI bound as a second-shell ligand of the tetrahedrally coordinated cobalt in Co(II)-substituted H64A HCA II, with 4-MI located about 4.5 A from the metal. Binding constants of 4-MI to H64A HCA II were estimated from: (1) NMR relaxation of the protons of 4-MI by Co(II)-H64A HCA II, (2) the visible absorption spectrum of Co(II)-H64A HCA II in the presence of 4-MI, (3) the inhibition by 4-MI of the catalytic hydration of CO2, and (4) from the catalyzed exchange of 18O between CO2 and water. These experiments along with previously reported crystallographic and catalytic data help identify a range of distances at which proton transfer is efficient in carbonic anhydrase II.     

Proton transfer within the active-site cavity of carbonic anhydrase III

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.     

Structure and catalysis by carbonic anhydrase II: role of active-site tryptophan 5

The tryptophan residue Trp5, highly conserved in the α class of carbonic anhydrases including human carbonic anhydrase II (HCA II), is positioned at the entrance of the active site cavity and forms a π-stacking interaction with the imidazole ring of the proton shuttle His64 in its outward orientation. We have observed that replacement of Trp5 in HCA II caused significant structural changes, as determined by X-ray diffraction, in the conformation of 11 residues at the N-terminus and in the orientation of the proton shuttle residue His64. Most significantly, two variants W5H and W5E HCA II had His64 predominantly outward in orientation, while W5F and wild type showed the superposition of both outward and inward orientations in crystal structures. Although Trp5 influences the orientation of the proton shuttle His64, this orientation had no significant effect on the rate constant for proton transfer near 1 μs⁻¹, determined by exchange of ¹⁸O between CO₂ and water measured by mass spectrometry. The apparent values of the pK_a of the zinc-bound water and the proton shuttle residue suggest that different active-site conformations influence the two stages of catalysis, the proton transfer stage and the interconversion of CO₂ and bicarbonate.     

Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II

The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹⁸O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had no effect on the interconversion of bicarbonate and CO₂, but in some cases caused enhancements in the rate constant of proton transfer by nearly 10-fold. The variant Y7I HCA II enhanced intramolecular proton transfer approximately twofold; its structure was determined by X-ray crystallography at 1.5 Å resolution. No changes were observed in the ordered solvent structure in the active-site cavity or in the conformation of the side chain of the proton shuttle His64. However, the first 11 residues of the amino-terminal chain in Y7I HCA II assumed an alternate conformation compared with the wild type. Differential scanning calorimetry showed variants at position 7 had a melting temperature approximately 8 °C lower than that of the wild type.     

Proton transfer to residues of basic pK(a) during catalysis by carbonic anhydrase.

The maximal velocity in the hydration of CO(2) catalyzed by the carbonic anhydrases in well-buffered solutions is limited by an intramolecular proton transfer from zinc-bound water to acceptor groups of the enzyme and hence to buffer in solution. Stopped-flow spectrophotometry was used to accumulate evidence that this maximal velocity is affected by residues of basic pK(a), near 8 to above 9, in catalysis of the hydration of CO(2) by carbonic anhydrases III, IV, V, and VII. A mutant of carbonic anhydrase II containing the replacement His-64-->Ala, which removes the prominent histidine proton shuttle (with pK(a) near 7), allows better observation of these basic groups. We suggest this feature of catalysis is general for the human and animal carbonic anhydrases and is due to residues of basic pK(a), predominantly lysines and tyrosines more distant from the zinc than His-64, that act as proton acceptors. These groups supplement the well-studied proton transfer from zinc-bound water to His-64 in the most efficient of the carbonic anhydrases, isozymes II, IV, and VII.     

Proton transport in carbonic anhydrase: Insights from molecular simulation

This article reviews the insights gained from molecular simulations of human carbonic anhydrase II (HCA II) utilizing non-reactive and reactive force fields. The simulations with a reactive force field explore protein transfer and transport via Grotthuss shuttling, while the non-reactive simulations probe the larger conformational dynamics that underpin the various contributions to the rate-limiting proton transfer event. Specific attention is given to the orientational stability of the His64 group and the characteristics of the active site water cluster, in an effort to determine both of their impact on the maximal catalytic rate. The explicit proton transfer and transport events are described by the multistate empirical valence bond (MS-EVB) method, as are alternative pathways for the excess proton charge defect to enter/leave the active site. The simulation results are interpreted in light of experimental results on the wild-type enzyme and various site-specific mutations of HCA II in order to better elucidate the key factors that contribute to its exceptional efficiency.     

Hydration of CO2 by carbonic anhydrase: intramolecular proton transfer between Zn2+-bound H2O and histidine 64 in human carbonic anhydrase II

The energy barrier for the intramolecular proton transfer between zinc-bound water and His 64 in the active site of human carbonic anhydrase II (HCA II) has been studied at the partial retention of diatomic differential overlap (PRDDO) level. The most important stabilizing factor for the intramolecular proton transfer is the zinc ion, which lowers the pKa of zinc-bound water and electrostatically repels the proton. The energy barrier of 127.5 kcal/mol for proton transfer between a water dimer is completely removed in the presence of the zinc ion. The zinc ligands, which donate electrons to the zinc ion, raise the barrier slightly to 34 kcal/mol for a 4-coordinated zinc complex including three imidazole ligands from His 94, His 96, and His 119 and to 54 kcal/mol for the 5-coordinated zinc complex including the fifth water ligand. A few model calculations indicate that these energy barriers are expected to be reduced to within experimental range (approximately 10 kcal/mol) when large basis set, correlation energies, and molecular dynamics are considered. The proton-transfer group, which functions as proton receiver in the intramolecular proton transfer, helps to attract the proton; and the partially ordered active site water molecules are important for proton relay function.     

Atomic crystal and molecular dynamics simulation structures of human carbonic anhydrase II: insights into the proton transfer mechanism

Human carbonic anhydrase II (HCA II) is a zinc-metalloenzyme that catalyzes the reversible interconversion of CO2 and HCO3-. The rate-limiting step of this catalysis is the transfer of a proton between the Zn-bound solvent molecule and residue His64. In order to fully characterize the active site structural features implicated in the proton transfer mechanism, the refined X-ray crystal structure of uncomplexed wild type HCA II to 1.05 A resolution with an Rcryst value of 12.0% and an Rfree value of 15.1% has been elucidated. This structure provides strong clues as to the pathway of the intramolecular proton transfer between the Zn-bound solvent and His64. The structure emphasizes the role of the solvent network, the unique positioning of solvent molecule W2, and the significance of the dual conformation of His64 in the active site. The structure is compared with molecular dynamics (MD) simulation calculations of the Zn-bound hydroxyl/His64+ (charged) and the Zn-bound water/His64 (uncharged) HCA II states. A comparison of the crystallographic anisotropic atomic thermal parameters and MD simulation root-mean-square fluctuation values show excellent agreement in the atomic motion observed between the two methods. It is also interesting that the observed active site solvent positions in the crystal structure are also the most probable positions of the solvent during the MD simulations. On the basis of the comparative study of the MD simulation results, the HCA II crystal structure observed is most likely in the Zn-bound water/His64 state. This conclusion is based on the following observations: His64 is mainly (80%) orientated in an inward conformation; electron density omit maps infer that His64 is not charged in an either inward or outward conformation; and the Zn-bound solvent is most likely a water molecule.     

Modelling the catalytic reaction in human aldose reductase

Aldose reductase (ALR2) has received considerable attention due to its possible link to long-term diabetic complications. Although crystal structures and kinetic data reveal important aspects of the reaction mechanism, details of the catalytic step are still unclear. In this paper a computer simulation study is presented that utilizes the hybrid quantum mechanical and molecular mechanical (QM-MM) potential to elucidate the nature of the hydride and proton transfer steps in the reduction of D-glyceraldehyde by ALR2. Several reaction pathways were investigated in two models with either Tyr48 or protonated His110+ acting as the potential proton donor in the active site. Calculations show that the substrate binds to ALR2 through hydrogen bonds in an orientation that facilitates the stereospecific catalytic step in both models. It is established that in the case that His110 is present in the protonated form in the native complex, it is the energetically favored proton donor compared with Tyr48 in the active pocket with neutral His110. The reaction mechanisms in the different models are discussed based on structural and energetic considerations.     

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.