Electrostatic control of proton-transfer reaction in the active site of carbonic anhydrase

Journal of Molecular Structure (Theochem) 582 (2002) 195±203

www.elsevier.com/locate/theochem

Electrostatic control of proton-transfer reaction in the active site of carbonic anhydrase A.N. Isaev* N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky pr. 47, 119991 Moscow, Russian Federation Received 9 May 2001; revised 9 October 2001; accepted 8 November 2001

Abstract The mechanism of proton-transfer (PT) in the active site (AS) of carbonic anhydrase was examined using ab initio calculations on geometry and electronic structure of model complex simulating PT system of the enzyme. The analysis of charge distribution on atoms of the complex gives evidence of the electrostatic control of the PT through H-bonded water chain connecting donor and acceptor groups. The PT reaction proceeds via the concerted mechanism in accordance with the principle of least dipole moment in transition state. The obtained results show that the electrostatic interaction in the enzyme AS can play the role of an `engine' driving the delivery of a proton from donor to acceptor through the bridge waters. The mobility of the water molecules forming a proton channel is found to be crucial to the PT process. q 2002 Elsevier Science B.V. All rights reserved. Keywords: H-bonds; Carbonic anhydrase; Proton-transfer complex; Electrostatic interaction; Dipole moment

1. Introduction Water molecules presented in enzymes active site (AS) can form channels for proton-transfer (PT) that connect reactive groups of enzyme and substrate. The geometry of proton channel may be such that it allows several water molecules oriented in a speci®c way, linked by H-bonds, to ®t in. Theoretical study of PT models, which mimic the H-bond network in enzyme AS, is useful in understanding the characteristics of PT in enzymatic systems. The PT system of carbonic anhydrase (CA), ef®cient catalyst of the reversible hydration of carbon dioxide, is the most interesting object for studying. Catalysis of the hydration of CO2 by CA occurs in two stages, the ®rst of which is conversion of CO2 into * Fax: 17-95-135-5328. E-mail address: [email protected] (A.N. Isaev).

HCO32 (Eq. (1)) by direct nucleophilic attack of the zinc-bound hydroxide on CO2 [1,2]. The second stage comprises the series of PT steps that regenerate the zinc-bound hydroxide (Eq. (2)). CO2 1 EZnOH2 1 H2 O , HCO2 3 1 EZnH2 O

…1†

His64-EZnH2 O 1 B , H1 His64-EZnOH2 1 B , His64-EZnOH2 1 BH1

…2†

B represents buffer in solution and His64 is a proton acceptor residue in CAII, which seems to ful®ll the criteria for a `perfectly evolved' enzyme [3], i.e. the most ef®cient catalyst possible for a particular reaction. Despite the general agreement about the chemical mechanism involved in the catalytic process, the details of the PT mechanism remain unresolved.

0166-1280/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0166-128 0(01)00786-2

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Fig. 1. Schematic diagram of PT complex simulating proton channel in AS of CA. Histidine bases are replaced by ammonia molecules.

Extensive experimental studies by Silverman and Lindskog [1] support the proposal of Steiner et al. [4] of an intramolecular proton shuttle between a Zn-bound water and the His64 residue in CAII. Since the distance between His64 and the zincbound water is too long for direct PT, there is likely to be proton conduction along intervening water molecules; indeed, a network of hydrogen-bonded waters has been observed in the crystal structure [5,6]. Studies have revealed that the Zn-bound water has about the same pKa (,7) as protonated His64. This similarity is rather important to the mutual proton exchange since a large difference in pKa could lead to a large barrier in one of the PT directions. In fact, this requirement has excluded the possibility of a PT role by other functional groups or residues [7]. Recent studies on isozymes CAIII (with Lys64) and CAV (with Tyr64) also support the PT mechanism outlined earlier [8]. Site-directed mutagenesis experiments showed that in both cases, placing a histidine at position 64 could increase the turnover number of the enzyme. The measurements also showed an increase of the maximal velocity of catalysis when Lys64 and Tyr64 are replaced by glutamic and aspartic acids, which can serve as ef®cient proton shuttle residues at pH 6±8. The imidazole ring of His64 may function, as indicated in Eq. (2), to transfer a proton from the AS to buffer in solution. Solvent hydrogen isotope experiments [4,9], and the release rate of 18O-labeled water

into solvent at different buffer concentrations [10], have shown that PT between Zn-bound water and His64 is the rate-limiting step of the maximal velocity at high buffer concentrations. At low buffer concentrations, the proton release into the medium becomes rate-limiting [11]. The basic issue is how the proton conduction along intervening water molecules occurs. What is the mechanism of proton translocation? Theoretical studies can help to resolve these issues. To date, there are a few theoretical works, in which the catalytic mechanism of CA was studied using quantum chemical methods [12±18]. The most reasonable values of the PT barrier were obtained by Lu and Voth [17] who carried out ab initio calculations of PT potential curves at various R(O´ ´´O) distances between donor and acceptor, and with different numbers of ligand water molecules. However, their calculations were performed at ®xed positions of the water molecules, with the assumption that PT through a water bridge proceeds step by step i.e. from Znbound water molecule to second water molecule and so on. Because the water molecules in enzyme AS possess translational and rotational mobility, the results obtained in Ref. [17] without geometry optimization do not throw light upon the mechanism of proton shuttle. To quantify the mechanism we performed ab initio calculations taking into account motion of all the protons and oxygen atoms of the water molecules forming PT channel [19]. In these calculations, concerted multiple transfer via a proton exchange by the bridge waters was found to be energetically preferable to single transfer. The obtained results lead us to conclusion that proton shuttle in the enzyme AS is a single-step process, which means that all H-bond protons are transferred from donor molecules to acceptor molecules in a concerted fashion. In this connection, a question of the force controlling such concerted process has a special interest. To answer this question is the primary goal of the present paper. This work has focused on a possible role of the Zn 21 dication placed near proton donor in the PT mechanism. The catalytic ef®ciency of enzymes is believed to result from the electrostatic stabilization of intermediates and transition states (TS's) in enzymes AS. The addressed question is whether the electrostatic interaction determines the pathway of

A.N. Isaev / Journal of Molecular Structure (Theochem) 582 (2002) 195±203

considered PT reaction? Positions of positive and negative charge centers attributed to the model PT complex were analyzed in order to describe how the charge distribution affects the dipole moment value and properties of the PT energy surface. 2. Details of calculations The PT complex simulating system with PT in the CAII AS is shown in Fig. 1. The ammonia molecules modeled the histidine residues here. The complex represents a structure where the Zn 21 ion is separated from the ultimate proton acceptor NH3 with three water molecules, which form the H-bonded chain of Z-con®guration. The ®rst of these waters binds directly to the Zn 21, and is the original proton donor. No restrictions were imposed on geometrical parameters of this structure upon optimization. The geometry of this (NH3)3Zn 21´ ´´OH2´ ´´OH2´´ ´OH2´´ ´NH3 system found in HF/6-311G calculations was used as a starting point at further consideration. Potential curves of PT were computed, after having chosen a starting point, by moving a given H atom in Ê along the H-bond axis from the uniform steps of 0.1 A donor to the acceptor, optimizing the remainder of the geometrical parameters at each step, at the HF/6-311G level. Such a pro®le provides an estimate of the position of the TS, more accurate structure of which was found using the QST3 procedure. These calculations provide also information on a change of the total dipole moment of the PT complex throughout the transfer process. Positions for centers of positive and negative charges 1 were derived at each step from Cartesian coordinates of atoms and from the values of their effective charges obtained in the HF/6-311G calculations according to Eq. (3) X q i zi i jˆ X ; …3† qi i

where qi is effective charge derived from the Mulliken population analysis and z i is coordinate Xi, Yi or Zi of ith atom de®ned in coordinate system related with the 1

Denoted as PC and NC centers below in the text.

197

21

Zn ion, summation is performed apart (i) for all 21 protons (with P regard to the charge on the Zn ion in the sum i qi ) and (ii) for all negatively charged atoms. Calculated in this way, j gives X, Y or Z coordinate of the PC and NC centers, correspondingly. The calculated coordinates were used to build displacement curves for the charge centers. The displacement curves show a change in the distance between the Zn 21 ion and charge centers with the PT. Single calculations on the PT complex were carried out at ®xed geometry of proton channel. A comparison of displacement curves calculated with and without optimization of the water molecules positions allows to analyze a role of structural ¯exibility of the H-bonded chain in the PT process. All the calculations were carried out on a SGI Power Challenge computer using the gaussian94 series of programs [20].

3. Results and discussion 3.1. Geometric and electronic structure of the PT complex and TS's The distance between Zn 21 ion and acceptor His64 residue and the distances of ligands around the Zn center can be derived from the enzyme X-ray structures. Here, we address to the work [21], where the pH-dependent structural variations of human CAII have been studied by X-ray crystallographic methods Ê resolution. The overall structure of CAII at at 2.3 A pH 6.5 or 5.7 was found to be quite similar to that determined at pH 8.5 in Ref. [5]. Transition metal binding sites in proteins are typically comprised of 3±4 protein ligands. In CA, the Zn 21 ion is coordinated with three histidine residues. It is important that the His ligands of Zn 21 would not have the pendant H atoms, so could not engage in the H-bond with the water bridge molecules. According to X-ray diffraction results [22], the Zn 21 ion is Ê from His64, leaving room for as located some 8 A many as three water molecules which could form a bridge to act as a proton shuttle. Our choice of the model PT complex was based on these speci®cs of the enzyme PT system. In terms of devising a suitable model, and one that is small enough to be treated by accurate ab initio calculations, it was deemed

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zation. Interatomic distances and effective charges on atoms and molecules calculated for the I and P structures are presented in Tables 1 and 2. Focusing attention on geometry of the PT complex, reported in Table 1, ultimate proton acceptor NH3 is Ê of the zinc, close to the value placed within 8±9 A derived from X-ray data of CAII. The chosen Zcon®guration of the water bridge prevents from the formation of side H-bonds by water molecules with model NH3 ligands. Regarding the complex between the Zn 21 ion and ligand molecules, the R(Zn±N) and R(Zn±O) bond lengths in our calculations agree with the results of X-ray diffraction experiments of CAII. For the protonated state of Zn-bound water, X-ray studies place the R(Zn±N) distances around 2.1± Ê , while R(Zn±O) is some 1.9±2.0 A Ê with a 2.3 A Ê [21,24]. It standard deviation of around 0.2±0.3 A is worth also to mention estimates obtained earlier with the use of other quantum chemical methods. Molecular mechanics re®nements of the solvated native CAI yielded a slightly distorted tetrahedron Ê , R(Zn±N) , 1.974 A Ê, at the zinc, with 1.936 A and R…Zn±O† ˆ 1:923 A [25]. Molecular dynamics simulations, using ligand bonding parameters derived from ab initio studies provides an average distance between the Zn 21 ion and the His resiÊ [26]. The comparison show dues of 2.10±2.15 A that further consideration of the PT system can be restricted to geometry of structures I and P as a

Table 1 Interatomic distances R in various protonation states of the PT complex. TS1 and TS2 refer to two possible TS's for the PT Ê) R (A

I

P

TS1

TS2

O1´´ ´O2 O2´´ ´O3 O3´´ ´N4 O1 ±H1 O2 ±H1 O2 ±H2 O3 ±H2 O3 ±H3 N4 ±H3 Zn±O1 Zn±N1 Zn±N2 Zn±N3 Zn´´´N4

2.495 2.574 2.783 1.010 1.486 0.986 1.588 0.975 1.809 1.965 2.092 2.091 2.091 8.662

2.666 2.643 2.616 1.696 0.973 1.670 0.974 1.560 1.056 1.860 2.128 2.116 2.116 8.896

2.499 2.372 2.660 1.483 1.018 1.129 1.243 1.001 1.659 1.892 2.123 2.111 2.111 8.538

2.423 2.427 2.507 1.053 1.370 1.065 1.362 1.290 1.217 1.943 2.094 2.095 2.095 8.258

adequate to replace the N-bearing His residues by the smaller ammonia molecules, a common substitution of imidazole ligands in such enzymatic studies [23]. We examined the PT complex in two protonation states relevant to the PT in the hydration direction from Zn-bound water molecule to ammonia acceptor molecule. These states correspond to the enzyme forms before PT (the initial state I with the protonated Zn-bound water) and after one (the transfer product P). Calculations on the PT complex in I and P states were performed with the complete geometry optimi-

Table 2 Effective charges Q on atoms and molecules (numbering the bridge water molecules shows direction from donor to acceptor) of the PT complex throughout the transfer process Q (a.u.)

I

P

TS1

TS2

Zn 21 Zn 21(NH3)3 O1 H1 O2 H2 O3 H3 N4 Water1 Water2 Water3 Acceptor ammonia

1.74 1.94 21.10 0.60 21.00 0.57 20.97 0.54 21.00 20.01 0.02 0.0 0.05

1.73 1.86 21.21 0.53 20.96 0.56 20.97 0.57 20.94 20.81 a 20.01 0.03 0.93 a

1.73 1.89 21.21 0.60 20.96 0.64 20.98 0.59 21.03 20.20 (20.80) b 0.16 (0.76) b 0.08 0.07

1.74 1.93 21.13 0.61 21.02 0.59 21.06 0.59 20.99 20.05 20.01 20.08 (20.67) b 0.21 (0.80) b

a b

The charges attributed to OH 2 and NH41 species. The charges in brackets take into account a displacement of H-bond proton in TS from donor to acceptor.

A.N. Isaev / Journal of Molecular Structure (Theochem) 582 (2002) 195±203

starting point and ®nal transfer product, respectively. In structure I, the two H-bonds between water Ê in length; molecules are found to be 2.50 and 2.57 A the R(O´ ´´N) distance of the last H-bond is equal to Ê . Shortening H-bonds as compared with the 2.78 A regular network in water is explained by the electrostatic effect pushing H-bond protons away from the Zn 21 ion. Indeed, the corresponding r(O´´ ´H) bond lengths of the three water molecules are 1.010, Ê , respectively, decreasing toward 0.986, and 0.975 A acceptor ammonia molecule. One can see that although some of these H-bonds are rather short, the bridging hydrogens are very unequally shared, clearly associated with their donating atom. The electrostatic interaction makes such structure more stable than one with center-symmetrical H-bonds. Other speci®cs of the structure I due to position of the Zn 21 ion and to dipole±dipole interaction between the molecules are (i) linear H-bonds characteristic for strong H-bonded complexes with bridging proton placed on a line between heteroatoms and (ii) Cs symmetry when heteroatoms of the H-bonded chain along with the side hydrogens of water molecules lie in one and the same plane with the Zn 21 ion. The plane of the PT complex symmetry determines position in this plane for the dipole moment vector. Both I and P are equilibrium structures corresponding to minima on the PT energy surface and the PT reaction can be described as a conversion I ! P. Due to concerted mechanism, the conversion I ! P may proceed through TS's of different structure, depending on H-bond proton (H1, H2 or H3), which initiates this conversion. Tables 1 and 2 show the change in structure of TS for the PT reaction as the identity of the proton determining conversion mode changes from H1 to H3. In lengthening the O1 ±H1 bond, when the H1 proton is moving along the H-bond axis between the O1 and O2 atoms, the reaction proceeds through the TS1 structure. In this case, the proton shared by the ®rst two waters along the chain was used as an `engine' to drive the process. That is, the r(O1´ ´´H1) Ê . For distance was lengthened in increments of 0.1 A each step along this transfer, the positions of the other two protons that are being transferred, H2 and H3, were optimized. The TS2 structure is found as TS in the case when the conversion I ! P results from lengthening the O3 ±H3 bond. In other words, TS1

199

and TS2 correspond with maximum on the PT potential curves calculated as functions of the distances r(O1´´ ´H1) and r(O3´´ ´H3), respectively. The details of the mechanism show that as a given proton is moved along, the other two protons tend to move along their respective paths as well, leading to what might be termed a concerted process, although the various PTs are not synchronous with one another. In terms of the positions of the three pertinent protons in the TS, their distances from the donor and acceptor atoms are reported in Table 1. The distances of H1, H2, and H3, from their respective donor atoms in TS1 are Ê , consistent with a concept 1.483 . 1.129 . 1.001 A of the proton conduction in which all three protons move in a generally concerted fashion, but that the proton nearest the Zn ion `pushes' the others along, and where the progress of the transfer of H2 lags behind H1, and H3 lags further behind the others. The lags in consecutive PT are found to be less for the TS2 structure. According to performed calculaÊ tions, H1 must stretch the O1 ±H1 bond to 1.483 A before reaching the TS for the transfer, whereas the Ê , r(O2´´ ´H2) stretch of the O3 ±H3 bond is only 1.290 A Ê , respectively, and r(O1´ ´´H1) are 1.065 and 1.053 A when H3 initiates the reaction. The series of lags is connected with a certain amount of charge buildup along the chain. For example, if both H1 and H2 protons in TS1 are considered to be associated with O2, due to their geometrical proximity, the resulting (H3O) 1 species is computed to have a Mulliken group charge of 10.76. A TS is traditionally de®ned as containing one imaginary frequency, the coordinate of which is associated with the transition from reactant to product. The analysis shows that the main contribution to the imaginary frequency of TS1 is derived from displacements of protons H1 and H2 such that both are moving along the conduction path. Other situation is found for the TS2 structure; here, the imaginary frequency is determined by displacement of the H3 proton toward acceptor ammonia molecule. 3.2. Electrostatic control of concerted PT It is seen from Tables 1 and 2 that the TS1 and TS2 geometric parameters and electronic structure differ considerably although both structures are TS's for one and the same PT reaction I ! P. One question

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A.N. Isaev / Journal of Molecular Structure (Theochem) 582 (2002) 195±203

then follows from the obtained data: What property of the TS1 and TS2 structures is a criterion for TS? In search of an answer to this question, we noticed the fact that dipole moment m of the PT complex in passing from the initial state I to the product P along the reaction coordinate is the least in TS. This fact leading thought that the mechanism of PT could be explained on the basis of analysis of the charge distribution on atoms. It seems clear that concerted change in coordinates of the H-bond protons during the PT process is accompanied by a displacement of the PC center in direction away from the Zn 21 ion. This displacement is to result in a change of the relative position of the PC and NC centers. Decreasing dipole moment of the PT complex in TS as compared with the initial state I gives evidence of approaching the PC and NC centers each other in structures TS1 and TS2. Fig. 2a and b show a change in distance RZn between the Zn 21 ion and the PC and NC centers with the increase of the interatomic distances r(O1´´ ´H1) and r(O3´ ´ ´H3). It is seen that RZn for the NC center decreases in TS. At the same time, a distance between the Zn 21 ion and the PC center increases so that displacement curves for the PC and NC centers come closer together as the reaction system moves to TS1 and TS2. The steep ascent of displacement curve for the PC center on passing TS results in the curves intersection, indicating jump of all H-bond protons to acceptors. What is the force controlling this process? The data of calculations prove clearly the electrostatic control of the PT reaction. Fig. 3 shows the fact that the dipole± dipole interaction between the molecules of the PT complex favors its stabilization. It is shown in Fig. 3 how the total dipole moment of the complex depends on the stretch of the O1 ±H1 and O3 ±H3 bonds (curves 1 and 2). In moving the system along the reaction coordinate, m decreases and amounts minimum value in TS. On passing the barrier, the increase of dipole moment due to a PT from Zn-bound water to acceptor ammonia provides electrostatic stabilization of the transfer product. The obtained results allow to propose the following hypothetical model of `the electrostatic engine'

201

driving PT. The work of the engine becomes clear from the scheme presented in Fig. 4. The scheme shows that the total dipole moment m of the PT complex can be considered as a vector sum of dipole moment derived from positions of the PC and NC centers and dipole moment attributed to hybrid sp3orbitals of heteroatoms (so-called `hybrid' dipole). The direction of the hybrid dipole is determined by orientation of unshared electron pairs toward the Zn 21 ion. Because the PT complex retains Cs symmetry as a proton is transferred, dipole moment vectors remain in one and the same plane throughout the transfer process. This fact allows applying 2D model of the evolution of the dipole moment to the process description. The charge centers approach in passing from the initial state to TS leads to a decrease of their contribution to the total dipole moment, and as a result m value decreases. The reaction system meets a criterion of TS at the distance between the PC and NC centers when m value of the PT complex becomes the least. As shown in Fig. 4, further decrease of the distance between the charge centers on passing the TS should lead to an increase of the total dipole moment that means electrostatic stabilization of the PT complex. It is clear that transfer of the H-bond protons to acceptor atoms becomes energetically pro®table, because one results in the formation of structure with the maximum dipole moment. According to the calculations, the charge centers lie nearly in line with the Zn 21 ion throughout the transfer process. For this reason, concerted PT is accompanied by anti-parallel reorientation of the dipole moment vector determined by positions of the charge centers. The dipole reorientation takes place at the point of intersection of the displacement curves, where positions of the PC and NC centers should practically coincide. Because the concerted PT reaction proceeds in accordance with the principle of least dipole moment in TS, the dynamics of H-bond oxygen atoms can play an extremely important role in this process. To analyze the role for translational and rotational motion of the water bridge molecules, we carried out calculations on

Fig. 2. Displacement of positive and negative charge centers throughout the PT process: (a) and (b) calculations with optimization of positions of the water molecules, the process is initiated by H1 proton (a) and by H3 proton (b); (c) calculations under the restriction that PT is not Ê. accompanied by the motion of oxygen atoms of the bridge waters. RZn and r(O±H) are indicated in units of A

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A.N. Isaev / Journal of Molecular Structure (Theochem) 582 (2002) 195±203

Ê ) between the proton undergoing Fig. 3. Dependence of the total dipole moment (D) of the PT complex on the interatomic distance r(O±H) (A the motion and donor oxygen atom. Cases presented by curves 1, 2 and 3 correspond to the same situations as (a), (b) and (c) in Fig. 2, respectively.

the PT complex with keeping orientation of the water molecules one and the same throughout the transfer process. In these calculations, the H-bond lengths and distance between the Zn 21 ion and terminal acceptor ammonia were kept ®xed and equal to their equilibrium values for the initial state I of the PT complex. The results of calculations are presented in Fig. 2c. There is no intersection of displacement curves showing positions of the PC and NC centers at ®xed orientation of the water molecules. In this case, the PC center is found closer to the Zn 21 ion then the NC center at any interatomic distance O1´´ ´H1 and dipole

moment of the PT complex decreases gradually with the increase of r(O1´ ´´H1) (curve 3 in Fig. 3). The `rigid' geometry of the proton channel prevents from achieving the criterion of the reaction TS. This means that the reaction system cannot overcome the barrier and concerted PT with the formation of the product P is forbidden. In rigid H-bonded chain, the H2 and H3 protons retain their positions near the oxygen atoms O2 and O3 when the proton H1 is placed near the acceptor O2 atom. This structure has no minimum on the PT energy surface. 4. Conclusions

Fig. 4. Scheme explaining how the electrostatic engine drives the conduction process. AB is a vector of dipole moment derived from positions of the positive and negative charge centers, AC is `hybrid' dipole vector determined by orientation of unshared electron pairs of heteroatoms toward the Zn 21 ion, the total dipole moment of the PT complex is de®ned as a vector sum AD of the AB and AC vectors. I, TS and P labels refer to the initial state, TS and transfer product, respectively.

The analysis of charge distribution in the PT complex clari®es the mechanism of proton conduction by a chain of water molecules in the AS of CA. The obtained results give evidence of the concerted mechanism of the PT reaction. The concerted PT proves to be possible due to electrostatic stabilization of the transfer product. TS of the PT reaction represents structure with the least dipole moment in passing from the initial state to the transfer product. The performed analysis allows an explanation of possible role for the water chain motion in electrostatic control of the PT process. As mentioned earlier, the considered system represents only a very small subset of the true enzyme,

A.N. Isaev / Journal of Molecular Structure (Theochem) 582 (2002) 195±203

including reduction of full amino acid residues to small model molecules. It is clear that electrostatic potential driving PT should depend on space positions of all ionic groups inside the enzyme AS. One can expect that geometry and orientation of proton channel is also very important for the PT process. On the question of the in¯uence on the process of the precise number of water molecules in the chain, the calculations performed in Ref. [19] suggest that there is not a great sensitivity here, and the chain can easily contain as many as ®ve and seven water molecules, with no negative implications for the process proceeding via proton exchange by the water molecules connecting donor and acceptor. The obtained results raise other important question: Is the electrostatic control of PT a certain general mechanism for enzymatic systems based on the interplay between the proton motion and displacement of the positive and negative charge centers? Further theoretical and experimental studies should give answer this question. Acknowledgements I wish to thank Prof. S. Scheiner for his continued interest in and promotion of this work. My thanks are due to the CACR Center of RAS for its support in providing access to computers. The calculations were performed under ®nancial support of the Russian Foundation for Basic Research. References [1] D.N. Silverman, S. Lindskog, Acc. Chem. Res. 21 (1988) 30. [2] D.W. Christianson, C.A. Fierke, Acc. Chem. Res. 29 (1996) 331. [3] J.R. Knowles, W.J. Albery, Acc. Chem. Res. 10 (1977) 105. [4] H. Steiner, B.H. Jonsson, S. Lindskog, Eur. J. Biochem. 59 (1975) 253.

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[5] A.E. Eriksson, T.A. Jones, A. Liljas, Proteins: Struct. Funct. Genet. 4 (1988) 274. [6] L.R. Scolnick, D.W. Christianson, Biochemistry 35 (1996) 16429. [7] K.M. Merz, J. Am. Chem. Soc. 113 (1991) 3572. [8] M. Qian, C. Tu, J.N. Earnhardt, P.J. Laipis, D.N. Silverman, Biochemistry 36 (1997) 15758. [9] Y. Poker, D.W. Bjorkquist, Biochemistry 16 (1977) 5698. [10] D.N. Silverman, C.K. Tu, S. Lindskog, G.C. Wynns, J. Am. Chem. Soc. 101 (1979) 6734. [11] S. Lingskog, G. Behravan, C. Engstrant, C. Forsman, B. Jonsson, Z. Liang, X. Ren, Y. Xue, in: F. Botre, G. Gros, B.T. Storey (Eds.), Carbonic Anhydrase: From Biochemistry and Genetics to Physiology and Clinical Medicine, VCH, Weinheim, 1991, p. 1. [12] J. Liang, W.N. Lipscomb, Biochemistry 27 (1988) 8797. [13] A. Vedani, D.W. Huhta, S.P. Jacober, J. Am. Chem. Soc. 111 (1989) 4075. [14] O. Jacob, O. Tapia, Int. J. Quant. Chem. 42 (1992) 1271. [15] Y.-J. Zheng, K.M. Merz, J. Am. Chem. Soc. 114 (1992) 10498. Ê qvist, A. Warshel, J. Mol. Biol. 224 (1992) 7. [16] A. A [17] D. Lu, G.A. Voth, J. Am. Chem. Soc. 120 (1998) 4006. [18] S. Toba, G. Colombo, K.M. Merz, J. Am. Chem. Soc. 121 (1999) 2290. [19] A. Isaev, S. Scheiner, J. Phys. Chem. B 105 (2001) 6420. [20] M.J. Frisch, G.W. Trucks, H.B. Schlegel, P.M.W. Gill, B.J. Johnson, M.A. Robb, J.R. Cheeseman, T. Keith, G.A. Peterson, J.A. Montgomery, K. Raghavachari, M.A. Al-Laham, V.G. Zakrzewski, J.V. Ortiz, J.B. Foresman, J. Cioslowski, B.B. Stefanov, A. Nanayakkara, M. Challacombe, C.Y. Peng, P.Y. Ayala, W. Chen, M.W. Wong, J.L. Andres, E.S. Replogle, R. Gomperts, R.L. Martin, D.J. Fox, J.S. Binkley, D.J. Defrees, J. Baker, J.P. Stewart, M. Head-Gordon, C. Gonzalez and J.A. Pople, gaussian94, Revision D.1, Gaussian Inc., Pittsburg, PA, 1995. [21] S.K. Nair, D.W. Christianson, J. Am. Chem. Soc. 113 (1991) 9455. [22] C.A. Lesburg, D.W. Christianson, J. Am. Chem. Soc. 117 (1995) 6838. [23] A. Pullman, D. Demoulin, Int. J. Quant. Chem. 16 (1979) 641. [24] K.K. Kannan, M. Ramanadham, T.A. Jones, Ann. N.Y. Acad. Sci. 429 (1984) 49. [25] A. Vedani, D.W. Huhta, S.P. Jacober, J. Am. Chem. Soc. 111 (1989) 4075. [26] Y.-J. Zheng, K.M. Merz, J. Am. Chem. Soc. 114 (1992) 10498.