Binding of Azide to Human Carbonic Anhydrase II: The Role

We report molecular dynamics simulations of the enzyme human carbonic anhydrase II (HCAII) inhibited by the azide anion (N3-) using a quantum ...
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J. Phys. Chem. 1996, 100, 17414-17420

Binding of Azide to Human Carbonic Anhydrase II: The Role Electrostatic Complementarity Plays in Selecting the Preferred Resonance Structure of Azide Kenneth M. Merz, Jr.* Department of Chemistry, 152 DaVey Laboratory, PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802

Lucia Banci Department of Chemistry, UniVersity of Florence, 50121 Florence, Italy ReceiVed: August 14, 1996X

We report molecular dynamics simulations of the enzyme human carbonic anhydrase II (HCAII) inhibited by the azide anion (N3-) using a quantum mechanical/molecular mechanical coupled potential. Experimentally, the azide ion binds to HCAII such that the structure of the active site is retained. This is interesting because the hydrogen bond interaction between the zinc-bound hydroxyl hydrogen and the hydroxyl oxygen of Thr199 in the native enzyme is replaced by what formally appears to be a repulsive interaction between the hydroxyl oxygen of Thr-199 and the zinc-bound azide nitrogen. Two possibilities were considered for this system: First, the binding of hydrogen azide (N3H) was considered, but we conclude based on experimental (i.e., pKas) and theoretical information that it is unlikely that this is what is bound to HCAII at physiological pH. When we bound the azide anion to the zinc ion in the HCAII active site we also found that the structure of the active site was retained. Upon further inspection, we determined that the reason for this has to do with the preferred azide resonance structure which placed extra positive charge on the central azide nitrogen, which allowed for favorable electrostatic interactions between the zinc-bound azide and the hydroxyl oxygen of Thr-199. This preferred enzyme resonance structure was 3.2 kcal/mol less stable than an alternative resonance structure in the gas phase when bound to zinc. Thus, HCAII is controlling the preferred resonance structure for the azide anion.

Introduction

SCHEME 1

Small anion inhibitors of human carbonic anhydrase II (HCAII) have generated a significant amount of controversy in the past few years due to the observation of unusual coordination modes.1-7 In particular, cyanide and cyanate have been problematic since both four-(solution phase studies)3 and fivecoordinate1 (X-ray studies) binding to the zinc ion in HCAII have been observed. To solve this problem, we suggested8 that HCAII hydrates hydrogen cyanide and hydrogen cyanate to form a five-coordinated complex, which is observed crystallographically.1 We also proposed that the solution phase studies were examining a four-coordinated species and that the difference between the solution phase and crystal phase structures had to do with differences in experimental conditions (low Vs high ionic strengths, etc.).8 Recently, Nair and Christianson9 and Jo¨nsson et al.10 have reported the X-ray structure of the azide-HCAII complex, which had several unexpected features. The structure is fourcoordinate, with one nitrogen bound to the zinc ion and the remaining nitrogen atoms placed into the A binding pocket of HCAII (Scheme 1). The zinc-bound nitrogen also forms a close contact with the hydroxyl oxygen of Thr-199 (3.3 Å), which suggests that the nitrogen atom has a bound proton. However, zinc-bound species (e.g., H2O) are known to have their pKas greatly reduced from their free solution values. For example, zinc-bound water suffers a >7pKa reduction when bound to a zinc ion. Free hydrogen azide has a pKa of ∼4.7,11 which, therefore, might also be expected to undergo a large pKa X

Abstract published in AdVance ACS Abstracts, October 1, 1996.

S0022-3654(96)02482-3 CCC: $12.00

reduction upon binding to the zinc ion in the active site of HCAII. Thus, it was concluded that azide was binding as the anion. Once bound as the anion to the HCAII active site, the azide ion could form a hydrogen bond with the hydroxyl hydrogen of Thr-199, but this would require that the hydrogen bond with Glu-106 be severed. From the crystal structure determination there was no evidence that this was the case.9,10 Hence, it was concluded that the zinc-bound azide nitrogen interacted with the hydroxyl oxygen of Thr 199 and that these close contacts with Thr-199 were not destabilizing.9 Moreover, this observation was given as evidence that Thr-199 is not the “gatekeeper”12 for the HCAII active site in that an anion binding to the zinc ion should be able to provide a proton to form a hydrogen bond with Thr-199. Finally, this observation was provided as © 1996 American Chemical Society

Binding of Azide to Human Carbonic Anhydrase II

J. Phys. Chem., Vol. 100, No. 43, 1996 17415

SCHEME 2

SCHEME 3

evidence favoring the “Lipscomb” mechanism for HCAII catalysis13 (see below) since this mechanism favors a binding mode for bicarbonate that does not involve a hydrogen bond with Thr-199. From a qualitative perspective, the conclusion that azide binds into the HCAII active site as the anion requires the terminal nitrogen of azide (which likely bears a partial negative charge) to form a repulsive interaction with the hydroxyl oxygen of Thr-199. Clearly, furthering our understanding of the azide-HCAII complex will have a large impact on our understanding of the role Thr-199 plays in the binding of inhibitors as well as on the catalytic function of HCAII. HCAII,14-16 one of seven isozymes of the zinc metalloprotein human carbonic anhydrase (HCA),17 is a 260 residue protein with a mass of ∼29 kDa. A single zinc atom is located in the enzyme active site and is necessary for catalytic activity.14-16 The active site itself lies at the bottom of a deep cavity (15 Å deep) in the protein, which is readily accessible to solvent.18 The active site cavity is divided into hydrophobic and hydrophilic regions, with a network of hydrogen-bonded water molecules connecting the active site region and the surrounding solvent environment.18 The catalytically necessary zinc atom lies at the bottom of the active site cleft and is tetrahedrally coordinated by three Histidine residues (His-94, -96, and -119) and a fourth ligand,18 whose identity is pH dependent. At high pH (>8) the fourth ligand is an hydroxide ion, while at acidic pH the fourth coordination site is occupied by a water molecule.14-16 The catalytic mechanism of HCAII has been studied in detail, yet much still remains unclear.14-16 Catalysis has been found to depend upon a group with a pKa of around 7, with the fourth zinc ligand (hydroxide/water) appearing to fulfill this requirement. This consideration, in conjunction with the observed ping-pong kinetics, gave rise to the mechanism shown in Scheme 2.16 The proton transfer step converting D into E has been implicated as the rate-limiting step at high concentration of external buffer, while E to A is thought to be rate limiting at low buffer concentrations.14-16 The conversion of D to A is kinetically distinct from the sequence of steps converting A into D, Via B and C. Although experiments have not completely elucidated the detailed structural changes in the mechanism for catalysis, there is considerable evidence that certain residues are catalytically important (Scheme 3). These include His-64, Glu-106, Thr-199, and several water molecules near the active site.14-16 Thr-199 is positioned with Thr-200 on the opposite side of the active site cavity from the zinc atom. These Thr residues, His-64 (located at the entrance of the active site cavity), and Glu-106 combine with other polar residues to constitute

the hydrophilic half of the cavity. Thr-199 is an important residue which is centered between the hydrophilic and hydrophobic halves of the cavity. It is locked into this position as part of a key hydrogen-bonding network. The hydrogen of the hydroxo/aquo zinc ligand is donated for hydrogen bonding to the γ-oxygen of Thr-199. The proton of the hydroxyl group on Thr-199 is then in turn donated to an -oxygen of Glu-106 forming a second hydrogen-bonding interaction (Scheme 3). The proximity of Thr-199 to the active site zinc atom and the rigidity of this hydrogen-bond network are considered to be crucial for catalysis and inhibitor binding.12,18 This group of hydrogen bonds may also play an important role in CO2 binding and catalysis. It has been suggested that this hydrogen-bond network serves to properly orient the lone pair electrons of the hydroxide ligand allowing for rapid addition to CO2, as the CO2 molecule approaches from the hydrophobic cavity (A binding site in Scheme 3).12,19,20 Bordering on this hydrogen-bond network is a group of eight water molecules which extends toward bulk water. It has been proposed that these water molecules serve to shuttle a proton out of the active site and into bulk solution Via His-64.14 As alluded to above, there is an alternative catalytic pathway to the Lindskog mechanism given in Scheme 2. In most of the details the two mechanistic schemes are essentially identical, but when it comes to the mode of binding of bicarbonate the two mechanisms differ. In this so-called Lipscomb mechanism13 the zinc bicarbonate form initially has the structure given as C1 where the carboxylate is pointing into the hydrophobic binding pocket labeled A in Scheme 3. This then undergoes a rearrangement to give the structure labeled C2 in Scheme 4 where the carboxylate group of the bicarbonate anion is bound directly to the zinc ion. The weakness in this mechanism is the requirement that a bicarbonate oxygen be in close proximity to the hydroxyl oxygen of Thr-199. This unfavorable electrostatic interaction presumably destabilizes this form of the bicarbonate complex to such an extent that it is not the favored bicarbonate binding mode.21 However, the suggestion that the azide anion can bind without forming a hydrogen-bonding interaction with the hydroxyl oxygen of Thr-199 has reopened this debate regarding the catalytic mechanism of HCAII.

17416 J. Phys. Chem., Vol. 100, No. 43, 1996 SCHEME 4

Merz and Banci SCHEME 5: Division of the Molecular System within a Combined Quantum Mechanical/Molecular Mechanical Calculationa

a The quantum mechanical (QM) region is surrounded by a molecular mechanical (MM) region and then by a boundary region (BR).

Computer simulation of proteins and other macromolecules has proven to be an invaluable aid in understanding their structural and dynamic properties.22-24 Because of the importance of understanding these systems, much effort has been directed toward refining the computational methods used to represent them. However, consideration of computational expense severely limits the degree of rigor which can be applied to any system. As a consequence of such considerations, molecular mechanical force fields are typically utilized in the study of macromolecular systems in solution.25-27 However, using such methods implicitly eliminates the possibility of studying inherently quantum mechanical events such as bond breaking or charge reorganization. Although methods have been proposed which incorporate terms for such events in an MM force field,28,29 these treatments will by their very nature be only an approximation to the quantum mechanical reality and often ignore the perturbing effect of the solvent and/or enzyme on the electronic structure of the reactive substrate. Other approaches use the so-called supermolecule approach and study enzymatic reactions in the gas phase.21,30-32 While this approach is computationally attractive and is capable of giving mechanistic insights, it is not capable of giving an accurate accounting of environmental effects that arise within an inhomogeneous environment like an enzyme active site.33-35 A method which potentially affords an escape from this difficulty is a quantum mechanical/molecular mechanical (QM/ MM) coupled potential originally pioneered by Warshel and Levitt34 and later utilized by others.36-40 In such a method, the computational model is partitioned into QM and MM regions (Scheme 5). Each portion is then treated using the appropriate computational method and communication between the two regions is faciliated Via van der Waal’s and electrostatic interactions. Using such a method it is thus possible to carry out a quantum mechanical examination of bond breaking/ forming events in a condensed phase (either solution or in an enzyme active site). The QM method to be employed may vary from empirical valence bond,24,41 semiempirical molecular orbital,36,37 density function,42,43 and even Hartree-Fock methods.39,44 For reasons of computational expense, NDDO-based semiempirical quantum mechanical methods have seen the most widespread use to date.37,38,45-51 However, it is important to note that there is nothing hindering the use of more sophisticated quantum mechanical treatments, other than the obvious demands placed upon CPU time. The utility of these methods for the study of quantum mechanical processes such as charge reorganization and chemical reactions in aqueous solution has been amply demonstrated by us52 and in the work of Karplus,45 Gao,47,50 and Warshel.24

While the use of QM/MM methodologies hold significant promise for the study of reactive events in solution, several comments are necessary about their application in enzyme systems. It is well-known that enzymes are very dynamic systems and that it is likely that the enzyme dynamics has an affect on the catalytic efficiency of the systems. Hence, it is critical that the dynamics of enzymes be considered when using QM/MM methodologies. For example, if energy minimization strategies are employed on going from a starting material to a product state, the enzyme structure does not have the opportunity to respond to the change in properties of the product state of the reaction. Hence, energy minimization strategies can lead to inaccurate results (i.e., energies, geometries, etc.). This important factor regarding the theoretical study of enzyme catalysis has been long recognized by Warshel and co-workers.24 Most of the early QM/MM studies on enzymes have utilized energy minimization strategies45,53 because these types of calculations are slightly more computationally expensive than traditional full MM studies. However, with the advent of faster workstations it is possible to carry out MD simulations using QM/MM methods and we have recently reported the first QM/ MM MD study of an enzyme system.52 In the present paper, we use the QM/MM approach in conjunction with MD simulations to probe the binding of the azide anion and hydrogen azide to HCAII. Computational Methods QM/MM Coupled Potential. The theoretical basis of the QM/MM method has already been extensively outlined in numerous publications so we will only comment upon some significant technical details of our implementation.34,36,43,54 The method we have developed couples together the MD program AMBER 4.055 and the semiempirical quantum mechanical program MOPAC 5.0. We have used standard AMBER force field parameters throughout,25,26 except for the active site region, where we have used the MM parameters of Hoops et al. developed especially for HCAII.56 Similar to the work of Karplus and co-workers36,45 and Singh and Kollman,39 we have used link atoms to cap exposed valence sites due to bonds which cross the QM-MM boundary. In this method the QM region of the system is treated as a closed-shell molecule with no exposed valence sites. In our system the imidazole rings are capped by hydrogen atoms at the C-γ carbon atom. These carbon atoms (three total) are then bonded to their respective C-β carbon atoms through molecular mechanical bonds (using a standard AMBER carbon-carbon parameter set) between the two carbon atoms. Finally, the nonbonded interactions between

Binding of Azide to Human Carbonic Anhydrase II

Figure 1. Zinc-hydrogen azide form of HCAII. Selected distances calculated from the MD trajectory are given in the figure.

the capping atoms and the remainder of the protein molecule are not evaluated. Computational Protocol. Starting coordinates for HCAII and crystallographically located water molecules were taken from the experimental crystal structure of Nair and Christianson.9 TIP3P57 water molecules were added, where possible, around the active site zinc atom to a distance of 15.0 Å using the EDIT module of AMBER 4.0.55 All residues were represented using the AMBER united atom model,25 except for the active site residues His-94, -96, and -119, which were represented as all atom residues. Minimization of all atoms was carried out for both forms of HCAII using an all-MM model and a standard version of AMBER 4.0. Following removal of bad contacts by MM minimization, a second minimization was carried out using a modified version of AMBER 4.0, in which the residues His-94, -96, and -119 and the active site zinc atom and its fourth ligand (azide) were treated as QM atoms using the PM3 Hamiltonian (for a total of 31 QM atoms).58-60 The junction between the QM and MM regions was made between C-β and C-γ of the His residues. In order to preserve integral charge in the MM region, the partial charges of the β-carbons of the QM His residues and the hydrogens attached to these carbons were changed to -0.080 and 0.048, respectively. Following the second minimization, MD simulations were carried out on the zinc-azide and zinc-hydrogen azide forms of HCAII. A 15 Å sphere was defined around the active site zinc atom, and only residues within this sphere as well as the cap water molecules were allowed to move during the MD simulations. The MD simulations covered 102 ps. The temperature was raised from 0 to 300 K during the first 6 ps of simulation, and the temperature was then maintained at 300 K by coupling to a constant temperature heat bath.61 A 10 Å nonbond cutoff distance was used and the nonbond pair list was updated every 25 time steps. The SHAKE algorithm was used to constrain all bonds between pairs of MM atoms.62 A 1 fs time step was employed during the MD simulations, and coordinates were saved for analysis every 25 (i.e., every 0.025 ps) time steps. Results and Discussion Zinc Hydrogen Azide Form of HCAII. We began our study by examining hydrogen azide bound to HCAII. Even though it was thought that HN3 would not bind to HCAII in its neutral form (because of the low pKa of HN3), we decided to study this system to determine if it was or was not able to form a stable complex with HCAII. The basic structure we examined is given in Figure 1.

J. Phys. Chem., Vol. 100, No. 43, 1996 17417

Figure 2. Fluctuations in the calculated heat of formation for the zinchydrogen azide form of HCAII over the last 96 ps (6-102 ps) of the MD simulation.

Figure 3. Fluctuations in the calculated heat of formation for the zincazide form of HCAII over the last 96 ps (6-102 ps) of the MD simulation.

From the simulations we have done on this system we found that the hydrogen azide form of HCAII is indeed stable and retains the general structure given in Figure 1. This is readily confirmed when one considers the average Zn-N bond distance (2.09 Å) given in Figure 1. Further distance analyses indicate that the N1-N2 bond distance is longer than the N2-N3 distance. We also found that the hydrogen bond between the azide hydrogen and OG1 of Thr-199 (2.12 Å) is retained throughout the MD simulation, which indicates that this is a fairly strong interaction. The terminal nitrogen (N3) has two weak hydrogenbond interactions with the main chain N-H of Thr-199 (3.17 Å) and Thr-200 (2.9 Å). All of these analyses indicate that hydrogen azide is snugly bound into the active site of HCAII and is, indeed, well suited to fit into the environment presented by HCAII. The fluctuations in the heat of formation for this system are given in Figure 2 (average ∆Hf ) 219.5 ( 22.0 kcal/mol (error bars are (1σ)), while that for the zinc-azide system is given in Figure 3 (average ∆Hf ) 157.0 ( 16.1 kcal/mol). While we have termed these values as heats of formation, it must be stressed that these values include environmental effects which alter the heat of formation values. Thus, these computed energies are probably better described as solvation enthalpies. If we assume that solvation entropy affects are small in this system (likely to be the case since the QM fragment in the enzyme active site is quite constrained by the environment), we can assume that our computed solvation enthalpy is of a similar magnitude as the solvation free energy of this system.

17418 J. Phys. Chem., Vol. 100, No. 43, 1996

Merz and Banci

Figure 4. Gas-phase heats of formation (kcal/mol) for the two possible resonance structures for the azide anion bound to a model of the HCAII active site. Atomic point charges calculated using electrostatic potential fitting methods are given in parentheses.

Figure 5. Zinc-azide form of HCAII. Selected distances and atomic point charges (obtained using electrostatic potential fitting methods) evaluated from the MD trajectory are given in the figure.

In order to roughly estimate the stability of the hydrogen azide form of HCAII to that of the azide form we still need to account for a proton. The proton ultimately ends up in bulk aqueous solution, so the heat of formation for H2O (-53.4 kcal/mol) and H3O+ (159.1 kcal/mol) along with their respective solvation free energies (-6.3 kcal/mol63 and -102 kcal/mol,64 respectively) can be used to estimate the stability of these two binding modes with respect to each other. The process we are considering can be given as

R3Zn2+N3H + H2O h R3Zn2+N3- + H3O+ 219.2 157.0 57.1 -59.7

(1)

From this analysis we estimate that the hydrogen azide form of the enzyme is less stable that the azide form by ∼54.6 kcal/ mol. This result along with the expectation that the pKa of hydrogen azide should be greatly reduced in the presence of HCAII demonstrates that it is likely that the azide anion is bound, and not hydrogen azide, to this enzyme.9,10 Zinc Azide Form of HCAII. Prior to the start of our MD simulations on the zinc-azide form of HCAII we calculated the gas-phase energies using PM3 for the active site model (i.e., (imidazole)3ZnN3) to determine the preferred resonance structure for this complex. The results of this calculation are given in Figure 4. From this gas-phase study we find that the resonance structure containing the triple bond is more stable than the structure that has two double bonds by 3.2 kcal/mol. We also calculated the PM3 electrostatic potential derived (ESP) charges65,66 for these two complexes and they are given in Figure 4. The experimental structure9 for the zinc-azide form of HCAII is given in Scheme 1 (experimental distances), while Figure 5 gives the average structure obtained from our MD simulations.

The first thing we note is that once the environmental effects are turned on, the preferred resonance structure is the resonance structure with two double bonds (compare Figures 4 and 5). Thus, the enzyme site is overcoming the intrinsic destabilization of this resonance structure over the one with a formal triple bond. This is no small effect (it is at least ∼3 kcal/mol) and demonstrates the inability of gas-phase calculations to capture the subtleties of how an enzyme can affect substrate structure. The reason for this difference in the observed resonance structure will be discussed further below. The calculated structure (Figure 5) is in reasonable accord with the experimental structure (Scheme 1) for the zinc-azide complex of HCAII, but some differences are present. The ZnN1 distance is ∼2.0 Å in both the experimental structure as well as the calculated average structure, but the N1-N2 (calcd 1.19 Å, expt 1.27 Å) and the N2-N3 (calcd 1.16 Å, expt 1.33 Å) distances vary between the two by ∼0.15 Å. The reason for this difference is not clear (e.g., potentially PM3 gives too short NdN bonds, etc.), but nonetheless they both predict that the resonance structure with two NdN bonds is favored. The distances between N1 and N2 to Oγ of Thr-199 are in good accord with experiment, but critically we found that the distance between N2 and Oγ (3.3 Å) is shorter than the distance between N1 and Oγ (3.6 Å). We also find that the Hγ proton from Thr199 is closer to N1 (3.55 Å) than is Oγ. The importance of these observations will become apparent below. The most significant differences arise when we look at the main chain N-H from Thr-199 and Thr-200 to N3 distances. The X-ray structure predicts that the Thr-199 N-H to N3 distance is ∼2.5 Å (assuming 1.0 Å for the N-H bond) and our calculated value is 2.32 Å, which is in good accord with experiment. However, the Thr-200 N-H to N3 distance is ∼4.4 Å, while we calculate this distance to be 2.65 Å. From our simulations we find that the carbonyl adjacent to the mainchain N-H of Thr-200 hydrogen bonds to the side chain of Arg-246. This causes the main chain NH of Thr-200 to tilt toward N3 of the azide ion. In the crystal structure neither of these interactions is observed. Thus, it appears that our simulations tend to overestimate the strength of these interactions. Nonetheless, most of the calculated details are in very good accord with experimental information. Why then does azide bind to HCAII as observed experimentally even when formal expectations would regard the interaction between two like charged species as being repulsive. The answer partially lies in the charge distribution of the azide anion in the presence of the enzyme. In Figure 5 we give the ESP charges of the zinc ion and the three nitrogen atoms of azide in the presence of the enzyme environment and in parentheses we give the ESP charges when the enzyme environment is turned off (i.e., a gas-phase calculation at the geometry generated during the MD simulation). All charges were averaged over the last 78 ps of the simulation (624 separate charge sets evaluated every 0.125 ps). From these charges we find that when the enzyme environment is turned on the charge on atom N2 becomes more positive by ∼0.12 charge units, while the charge on N1 becomes less negative by ∼0.03 charge units. Finally, N3 has its charge increased from -0.72 to -0.96 or a 0.24 increase in net negative charge, while the zinc ion becomes more positive by 0.29 charge units. Thus, by increasing the positive charge on N2 the hydroxyl oxygen of Thr-199 can favorably interact electrostatically with N2 and by decreasing the net negative charge on N1 the repulsive electrostatic interaction with the hydroxyl oxygen of Thr-199 is reduced. Moreover, the net negative charge on N1 interacts favorably with the positive charge on the hydroxyl of Thr-199. It is interesting that N3 undergoes the largest charge

Binding of Azide to Human Carbonic Anhydrase II shift of all the nitrogen atoms, but this is facilitated by the fact that this nitrogen can interact readily with several main chain N-H groups from Thr-199 and Thr-200. Comparing the simulated results (Figure 5) to the gas-phase results (Figure 4), we find that most of the charges are enhanced in the enzyme relative to the gas-phase charges. The reason for this arises from polarization effects as well as geometric (i.e., the gasphase geometry and the geometries from the MD simulations differ) effects. The net result of all these changes in the charge distribution on azide is to allow this molecule to readily interact with the active site of HCAII in an electrostatically favorable manner. Conclusions From our simulations on the zinc-azide and zinc-hydrogen azide form of HCAII we have observed that the latter form is unstable relative to the former form of this enzyme. Moreover, we find that the active site of HCAII selectively favors one resonance structure of the azide anion over another that is actually the preferred form in the gas phase. This control of the preferred resonance form of a small molecule binding to an enzyme is an interesting observation and points to the fact that gas-phase calculations on enzyme activity can be very misleading if not carefully interpreted. It has been suggested that the azide complex demonstrates that Thr-199 is not the gatekeeper12 of the HCAII active site.9 Our calculated results do not support this conclusion because the azide ion is responding to the presence of the side chain of Thr-199 by adopting a resonance structure that can form favorable electrostatic interactions with this residue. Originally, the gatekeeper idea required that a hydrogen bond was present between zinc-bound species and Thr-199.12 This is not the case for azide, but hydrogen bonds are electrostatic in nature and the azide-Thr 199 interactions we are a proposing are also electrostatic in nature. Thus, Thr-199 is still controlling the outcome of events occurring around the zinc ion within the HCAII active site. Moreover, the azide complex of HCAII does not provide support for the Lipscomb mechanism of HCAII catalysis because Thr-199 is still playing a critical role in determining what is occurring within the HCAII active site. Thus, the bicarbonate ion is going to have to acknowledge the presence of Thr-199 in some electrostatically complementary manner and it is not clear at this point how the Lipscomb mechanism accomodates this.67 Bromide ion also binds to HCAII in such a manner that the zinc-bound bromine atom is in proximity with the hydroxyl oxygen of Thr-199 (Br- - -Thr-199 Oγ distance is 3.64 Å).10 It was also observed that the zinc-bromide complex is distorted away from the normal tetrahedral geometry observed in HCAII.10 It has been argued that because bromine is a polarizable anion this minimizes the unfavorable interaction with the hydroxyl oxygen of Thr 199.10 Moreover, since zinc is a soft cation and bromide is a soft anion the expected affinity for these two ions would be expected to be favorable (relative to a case where there is a hard/soft mismatch). Another factor that plays a role is the hydrogen bond between Thr-199 and Glu-106 which likely is strong enough to retain the positioning of the hydroxyl group of Thr-199. Thus, for azide it appears that electrostatic complementarity plays a role in binding this anion, but bromide on the other hand represents an exception to this rule. In conclusion, we have demonstrated the power of using a QM/MM approach to aid in our understanding of enzyme structure, function, and dynamics and this approach is sure to continue to advance our understanding of not only enzymes but other biological processes.

J. Phys. Chem., Vol. 100, No. 43, 1996 17419 Acknowledgment. We thank the NIH for supporting this research through Grant GM44974. NATO also provided support for L.B. and K.M.M. to jointly work on this project and they are also acknowledged. We also thank the Pittsburgh Supercomputer Center and the Cornell Theory Center for generous allocations of supercomputer time. References and Notes (1) Lindahl, M.; Svensson, L. A.; Liljas, A. Proteins 1992, 15, 177192. (2) Ha˚kansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol. 1992, 227, 1192-1204. (3) Bertini, I.; Luchinat, C.; Pierattelli, R.; Vila, A. J. Inorg. Chem. 1992, 31, 3975-3979. (4) Bertini, I.; Canti, G.; Luchinat, C.; Scozzafava, A. J. Am. Chem. Soc. 1978, 100, 4873-4877. (5) Banci, L.; Bertini, I.; Donaire, A.; Luchinat, C.; Martinez, J. M.; Moratal, J. M. Comm. Inorg. Chem. 1990, 9, 245-261. (6) Banci, L.; Bertini, L.; Luchinat, C.; R., M.; Moratal Mascarell, J. M. Gazz. Chim. Acta. 1989, 119, 23-29. (7) Bertini, I.; Luchinat, C.; Scozzfava, A. Struct. Bonding (Berlin) 1982, 48, 45-91. (8) Peng, Z.; Merz, K. M., Jr.; Banci, L. Proteins 1993, 17, 203-216. (9) Nair, S. K.; Christianson, D. W. Eur. J. Biochem. 1993, 213, 507515. (10) Jo¨nsson, B. M.; Hakansson, K.; Liljas, A. FEBS Lett. 1993, 322, 186-190. (11) Christensen, J. J.; Hansen, L. D.; Izatt, R. M. Handbook of Proton Ionization Heats and Related Quantities; John Wiley and Sons: New York, 1976. (12) Xue, Y.; Liljas, A.; Jonsson, B.-H.; Lindskog, S. Proteins 1993, 17, 93-106. (13) Liang, J.-Y.; Lipscomb, W. N. Int. J. Quantum Chem. 1989, 36, 299-312. (14) Silverman, D.; Vincent, S. H. CRC Crit. ReV. Biochem. 1983, 14, 207-255. (15) Silverman, D. N.; Lindskog, S. Acc. Chem. Res. 1988, 21, 30-36. (16) Lindskog, S. In Zinc Enzymes; Spiro, T. G., Ed.; Wiley: New York, 1983; pp 77-121. (17) Tashian, R. E. BioEssays 1989, 10, 186-192. (18) Eriksson, A. E.; Jones, A. T.; Liljas, A. Proteins 1988, 4, 274282. (19) Merz, K. M., Jr. J. Mol. Biol. 1990, 214, 799-802. (20) Merz, K. M., Jr. J. Am. Chem. Soc. 1991, 113, 406-411. (21) Zheng, Y. J.; Merz, K. M., Jr. J. Am. Chem. Soc. 1992, 114, 1049810507. (22) McCammon, J. A.; Harvey, S. C. Dynamics of Proteins and Nucleic Acids; Cambridge University Press: New York, 1987. (23) Brooks, C. L., III; Karplus, M.; Pettit, B. M. Proteins: A Theoretical PerspectiVe of Dynamics, Structure, and Thermodynamics; John Wiley and Sons: New York, 1988; Vol. LXXI. (24) Warshel, A. Computer Modelling of Chemical Reactions in Enzymes and Solutions; John Wiley & Sons, Inc.: New York, 1991, pp 236. (25) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.; Alagona, G.; Profeta, S.; Weiner, P. J. Am. Chem. Soc. 1984, 106, 765784. (26) Weiner, S. J.; Kollman, P. A.; Nguyen, D. T.; Case, D. A. J. Comput. Chem. 1986, 7, 230-252. (27) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983, 4, 187-217. (28) Dang, L. X.; Rice, J. E.; Caldwell, J.; Kollman, P. A. J. Am. Chem. Soc. 1991, 113, 2481-2486. (29) Sprik, M.; Klein, M. L. J. Chem. Phys. 1988, 89, 7556-7560. (30) Jacob, O.; Cardenas, R.; Tapia, O. J. Am. Chem. Soc. 1990, 112, 8692-8705. (31) Jacob, O.; Tapia, O. Int. J. Quantum Chem. 1992, 42, 1271-1289. (32) Merz, K. M., Jr.; Hoffmann, R.; Dewar, M. J. S. J. Am. Chem. Soc. 1989, 111, 5636-5649. (33) Tapia, O.; Johannin, G. J. Chem. Phys. 1981, 75, 3624-3635. (34) Warshel, A.; Levitt, M. J. Mol. Biol. 1976, 103, 227-249. (35) Tapia, O.; Lamborelle, C.; Johannin, G. Chem. Phys. Lett. 1980, 72, 334-341. (36) Field, M. J.; Bash, P. A.; Karplus, M. J. Comput. Chem. 1990, 11, 700-733. (37) Luzhkov, V.; Warshel, A. J. Comput. Chem. 1992, 13, 199-213. (38) Gao, J. J. Phys. Chem. 1992, 96, 537-540. (39) Singh, U. C.; Kollman, P. A. J. Comput. Chem. 1986, 7, 718730. (40) A° qvist, J.; Warshel, A. Chem. ReV. 1993, 93, 2523. (41) A° qvist, J.; Warshel, A. J. Mol. Biol. 1992, 224, 7-14. (42) Stanton, R. V.; Hartsough, D. S.; Merz, K. M., Jr. J. Phys. Chem. 1993, 97, 11868-11870.

17420 J. Phys. Chem., Vol. 100, No. 43, 1996 (43) Stanton, R. V.; Hartsough, D. S.; Merz, K. M., Jr. J. Comput. Chem. 1994, 16, 113-128. (44) Stanton, R. V.; Merz, K. M., Jr., unpublished results. (45) Bash, P. A.; Field, M. J.; Davenport, R. C.; Petsko, G. A.; Ringe, D.; Karplus, M. Biochemistry 1991, 30, 5826-5832. (46) Gao, J.; Pavelites, J. J. J. Am. Chem. Soc. 1992, 114, 1912-1914. (47) Gao, J.; Xia, X. Science (Washington, D.C.) 1992, 258, 631-635. (48) Gao, J. J. Phys. Chem. 1992, 96, 537-540. (49) Gao, J. J. Phys. Chem. 1992, 96, 6432-6439. (50) Gao, J.; Xia, X. J. Am. Chem. Soc. 1993, 115, 9667-9675. (51) Gao, J. Int. J. Quantum. Chem: Quantum. Chem. Symp. 27 1993, 491-499. (52) Hartsough, D.; Merz, K. M., Jr. J. Phys. Chem. 1995, 99, 1126611275. (53) Lyne, P. D.; Mulholland, A. J.; Richards, W. G. J. Am. Chem. Soc. 1995, 117, 11345-11350. (54) Thompson, M. A.; Schenter, G. K. J. Phys. Chem. 1995, 99, 63746386. (55) Pearlman, D. A.; Case, D. A.; Caldwell, J. C.; Seibel, G. L.; Singh, U. C.; Weiner, P.; Kollman, P. A. In University of California, San Francisco: 1991. (56) Hoops, S. C.; Anderson, K. W.; Merz, K. M., Jr. J. Am. Chem. Soc. 1991, 113, 8262-8270.

Merz and Banci (57) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J.; Impey, R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926. (58) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 209-220. (59) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 221-264. (60) Stewart, J. J. P. J. Comput. Chem. 1991, 12, 320-341. (61) Berendsen, H. J. C.; Potsma, J. P. M.; van Gunsteren, W. F.; DiNola, A. D.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684-3690. (62) van Gunsteren, W. F.; Berendsen, H. J. C. Mol. Phys. 1977, 34, 1311. (63) Ben-Naim, A.; Marcus, Y. J. Chem. Phys. 1984, 81, 2016-2027. (64) Marcus, Y. Ion SolVation; John Wiley: New York, 1985. (65) Merz, K. M., Jr. J. Comput. Chem. 1992, 13, 749-767. (66) Besler, B. H.; Merz, K. M., Jr.; Kollman, P. A. J. Comput. Chem. 1990, 11, 431-439. (67) Merz, K. M., Jr., unpublished results. Preliminary studies of the HCAII-bicarbonate complex indicate that even in the Lipscomb-like binding mode there is a hydrogen bond between Thr-199 and zinc-bound bicarbonate.

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