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Theoretical Studies on the Catalytic Cycle of Histidine Acid Phosphatases Revealing an Acid Proof Mechanism Hao Zhang, Ling Yang, Wanjian Ding, and Yingying Ma J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04808 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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The Journal of Physical Chemistry

Theoretical Studies on the Catalytic Cycle of Histidine Acid Phosphatases Revealing an Acid Proof Mechanism Hao Zhang*a, Ling Yangb, Wanjian Dingc, Yingying Ma*d,e a

College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030,

P.R. China b

MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and

Storage, School of Chemistry and Chemical Engineering, Harbin Institute of Technology 150080, P.R. China c

College of Chemistry, Beijing Normal University, Beijing100086, P.R. China

d

Institute of Mining Technology, Inner Mongolia University of Technology. Hohhot 010051,

P. R. China e

Inner Mongolia Key Laboratory of Theoretical and Computational Chemistry Simulation.

Hohhot 010051, P. R. China * Corresponding authors. Hao Zhang, Email: [email protected]; Yingying Ma, Email: [email protected]

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ABSTRACT After reporting the mechanisms for purple acid phosphatases against acid environments and alkaline phosphatases against alkaline environments, in the present work, we continued investigating the relationship between catalytic structures of histidine acid phosphatases (HAPs) and acid environments. Based on the comparison of the crystal structures of several HAP members, a series of models were constructed and calculated using density functional theory. Our calculations describe a complete catalytic cycle for HAPs including a free stage and a catalytic reaction stage. This cycle reveals a definite mechanism for HAPs to survive in acidic environments, which can be used to nicely interpret acidic pH optima of HAPs. It also suggests a free water molecule from solvent should be the nucleophile for hydrolyzing the phosphohistidine intermediate. Our studies are focused on the biological significance of enzymatic mechanisms and raise two concrete criteria: the logic-complete catalytic cycle, and the evolutional relation with family members and molecular environments.

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INTRODUCTION The enzyme structure-function studies, based on structural comparisons of evolutionarily related enzymes, can provide more general insights into enzymatic functions, such as identifying the role of catalysis related residues, and helping understand the evolution of enzymes for new catalytic functions.1-7 Until now, most studies on the enzymatic mechanisms still focus on an individual enzyme rather than on enzyme families and provide insights on enzymatic functions that are too limited. Our investigations have long been devoted to the evolution of the enzymatic mechanisms.8-11 On the one hand, the relationship of the catalytic mechanisms and the environments was a focus;9,10 on the other hand, the development of enzymatic activity in a family was also a concern.8,11 We have reported the mechanisms for purple acid phosphatases (PAPs) against acid environments9 and alkaline phosphatases (APs) against alkaline environments (Fig. 1).10 PAPs and APs all specialize in different active-site structures to adapt to different pH environments. In the present work, we continue investigating the relationship between the catalytic structures of histidine acid phosphatases (HAPs) and acid environments.

Fig. 1. Buffer mechanisms for PAPs against acid environments and E. coli AP against 3



alkaline environments. H+ and OH- in red represent exogenous proton and hydroxyl ions from solution. HAPs catalyze phosphoryl transfer from phosphomonoesters to water optimally at acidic pH,12 hydrolyzing a wide range of alkyl and aryl orthophosphate monoesters. Members in the HAP family have different optimum pH, such as the human prostatic acid phosphatase (HPAP) with a pH optima at about 5,13-15 Escherichia coli and the Aspergillus niger acid phosphatases (ECAP and ANAP) with a pH optima at 2.5,16,17 and the phytate from Aspergillus niger (ANPhyt) with two pH optima at 2.5 and 5.5 for the special substrate phytate.17-19 ECAP has been studied as a prototype for the common catalytic mechanism of the HAP family,16 which also reveals a highly conserved active site constituted of an aspartate, two histidines and three arginines. HAPs are classified as histidine phosphatases,20,21 attributed to their catalysis of dephosphorylation through a nucleophilic histidine. On the basis of crystallographic, kinetic and biochemical data, the dephosphorylation of phosphate monoesters catalyzed by HAPs was proposed to proceed in two reaction steps (Fig. 2).16,22,23 For the first step, a histidine residue performs an in-line nucleophilic attack on the P–O ester bond to form a covalent phosphohistidine intermediate; then, for the second step, the phosphohistidine is hydrolyzed by the attack of a water molecule to liberate phosphate, resulting in the formation of a noncovalent enzyme-inorganic phosphate complex. A series of computational studies by Sharma et al. support this mechanism.24-26

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Fig. 2. Reaction mechanism of HAPs for the first step (left) and the second step (right). The proton and the water molecule in red indicate their sources are undetermined. A complete catalytic cycle is the basis for understanding enzymatic function from the catalytic mechanism perspective. However, some details of the HAPs catalytic mechanism remain elusive (Fig. 2), which are indispensable to the catalytic cycle of HAPs. First, the ester oxygen atom of the leaving group should be protonated at the first step reaction, but it is not determined which is the proton donor.23 Second, it is not determined whether the water for hydrolyzing the phosphohistidine is from solvent or a specialized one at the second step reaction. In HPAP, a water molecule held in position by Asp258 and Arg15 was proposed to be the likely nucleophile attacking the phosphohistidine intermediate.14,21,27 Third, the free stage of enzymes before substrate binding has always been ignored, but it was indicated to be important for the regeneration of enzyme activity and some catalytic properties in our previous studies.9,10 In this study, the crystal structures of the HAP members have been compared, which brings important clues for the details of the HAP catalytic mechanism. Then, a series of models were constructed and calculated by using density functional theory (DFT) and B3LYP functional. The theoretical study was carried out to investigate the catalytic cycle of HAPs. This catalytic cycle is composed of a free stage and a catalytic reaction stage, including the catalytic mechanism details mentioned above. Meanwhile, a definite mechanism for HAPs to adapt acid environments was also proposed. 5



METHODS All calculations in this study were performed by using the DFT method with the B3LYP functional28-30 and the 6-31G(d, p) basis set. The frequency calculations were performed to obtain zero-point energies (ZPE) and to confirm the nature of the stationary points and including transition states along the reaction profiles. The polarization effects of the enzyme environments were evaluated by performing single-point calculations on the optimized structures at the same theory level as the geometry optimizations using the conductor-like polarizable continuum model (CPCM) method.31-34 The dielectric constants were set to four and eighty (ԑ=4 and 80), which are usually used in modeling the protein surroundings and the aqueous solution, respectively. All calculations were performed with the Gaussian 09 program package.35 The optimized geometry figures were created by the ChemCraft program.36 The energies were confirmed by the single-point calculations with the 6-311++G(2d,2p) basis set, and by dispersion-corrected density functional theory (DFT-D) calculations,37 which were supplied in supporting information. In all the models, the residues were truncated such that, in principle, only the side chains were retained. To keep the optimized structures close to the experimental structures, the truncation atoms were kept fixed at their X-ray positions during the geometry optimizations. Hydrogen atoms were added manually, and the truncated bonds were saturated by hydrogen atoms. The carbonyl of a glycine, well-conserved in HAPs (Gly13 in HPAP, Gly60 in ANAP and Gly64 in ANPhyt), makes an important hydrogen-bond with the catalytic histidine side chain holding it in an appropriate conformation for catalysis.21 Thus, the carbonyl of Gly9 and the hydrogen-bond with the catalytic histidine were considered in all the models. The 6


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un-catalytic histidine, also conserved in HAPs (His257 in HPAP, His318 in ANAP, and His338 in ANPhyt) was considered to be positively charged, as in the studies by Sharma et al..24-26 The pKa values for the residues in the crystal structures of ANAP (PDB code: 1qfx),17 ANPhyt (PDB code: 1ihp),38 and HPAP (PDB code: 1nd6)14 were calculated using PROPKA3.039-41 on the PDB2PQR service.42 RESULTS AND DISCUSSION PDB survey Although HAP members possess a highly conserved active site, as shown in Fig. 3, there are still some typical differences in their second shell residues of the active sites in HPAP,14 ANAP17 and ANPhyt.38 First, in HPAP, the un-catalytic histidine His257 is paired with Thr75 via hydrogen bond, while in ANAP and ANPhyt, the equivalent His318 and His338 are paired with Glu184 and Glu179, respectively. Second, in HPAP Asp179 connects with Arg15 via double hydrogen bonds, while no residues connect with the equivalent arginines Arg66 and Arg62 in ANAP and ANPhyt. Third, in ANAP and ANPhyt the aspartate residues Asp319 and Asp339 connect Tyr276 and Lys278 via a hydrogen bond, respectively, which is absent in HPAP.

Fig. 3. Active sites in the crystal structures of ANAP (PDB code: 1qfx),17 ANPhyt (PDB code: 1ihp),38 and HPAP (PDB code: 1nd6).14 The dashes show the potential hydrogen-bonds and 7



the important distances are in angstroms. The red arrows indicate the differences in the active-site structures. These figures were created with the software Pymol.43 Three water molecules (Wat1, Wat2 and Wat3) were found in the center of the active sites of the free enzymes ANAP and ANPhyt without substrate binding (Fig. 3a and b). Although no crystal structures for the free stage of HPAP have been resolved, two water molecules (WatA and WatB) were observed beside the product phosphate in HPAP (Fig. 3c). The WatA, held in position by Asp258 and Arg15, was suggested to be the likely nucleophile for hydrolyzing the phosphohistidine intermediate.14,21,27 It is worthy to mention that there is a general misconception for the hydrophobicity of the active pocket. The higher hydrophobicity of the active pocket was sometimes misinterpreted as the active pocket being absolutely exclusive of water molecules. This misconception often leads directly to the neglect on the crystal structures of free stage enzymes. In fact, the higher hydrophobicity should be a strict admittance constraint for water molecules entering the active pockets. Thus, when some water molecules can be always observed appearing in the active sites of crystal structures, there would be some significance of the catalytic structure and function for these water molecules.9,10 Free stage of enzyme To investigate the free stage of enzyme before substrate binding, the models were designed on the basis of the crystal structures of ANAP and ANPhyt (PDB codes: 1qfx17 and 1ihp,38 respectively). All the residues and the three water molecules (Wat1, Wat2 and Wat3), as shown in Fig. 3a and b, were considered in the models. After geometry optimizations, it was found that only when the three arginines were all deprotonated and sharing protons with the 8


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water molecules via hydrogen bonds, the models could maintain right conformations and hydrogen bonds as in the crystal structures (Fig. 4 and S1). The corresponding optimized geometries were named Free_ANAP and Free_ANPhyt models, respectively.

Fig.

4.

Optimized

geometries of

Free_ANAP,

Free_HPAP,

Free_HPAP_A

and

Free_HPAP_B models. The dashes show the potential hydrogen bonds and the important distances are in angstroms. The fixed atoms are marked with asterisks. Although no crystal structures for the free stage of HPAP have been resolved, for their highly conserved active sites, the active site structure of HPAP free stage should be similar to ANAP and ANPhyt. Therefore on the basis of the crystal structures of HPAP (PDB codes: 1nd6),14 the corresponding model was constructed by introducing the three water molecules (Wat1, Wat2 and Wat3) into HPAP. After geometry optimizations, it was found that only 9



when the two arginines, Arg79 and Arg11, were unprotonated and the one arginine Arg15 was protonated, the conformation and the hydrogen bonds of the model (named as Free_HPAP) could be maintained similar to the Free_ANAP model (Fig. 4). As we mentioned in the PDB survey above, two water molecules (WatA and WatB) were observed beside the product phosphate in HPAP (Fig. 3c). When WatA and WatB were successively added into the Free_HPAP model, they all result in more choreographed hydrogen-bond networks, as shown in Fig. 4. The corresponding optimized geometries were named as Free_HPAP_A and Free_HPAP_B models, respectively. Our calculations showed that in Free_ANAP and Free_ANPhyt models, there were three unprotonated and neutrally charged arginines. The arginine is a basic amino acid (pKa is ca. 12.5 in the general case), and its side chain guanidyl has strong ability of attracting protons. Thus, the three arginines, via water-mediated interactions, can attract protons from each other, which leads to the three arginines becoming the unusual deprotonated state and sharing protons with the water molecules in the models. Such observations are supported by the results of the pKa predictions by PROPKA3.0 (Table 1). In both the crystal structures of ANAP and ANPhyt, two of the three arginines of the active site gain the lowest and unusual pKa values among all arginine residues. In ANAP, the pKa values of Arg58 and Arg142 were calculated to be 10.26 and 10.97, respectively, while in ANPhyt, the pKa values of Arg66 and Arg156 were 8.62 and 10.97, respectively. Table 1. pKa values of arginines in ANAP, ANPhyt, and HPAP calculated by PROPKA3.0. The three arginines in the active sites are in red. The highest values are underlined. Three more values after Arg420 in ANAP are hidden in the table for alignment (Arg423 12.38, 10


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Arg428 12.42, and Arg434 12.09). ANAP

ANPhyt

HPAP

ARG49 12.28 ARG58 10.26 ARG62 12.36 ARG123 15.79 ARG129 12.20 ARG136 17.30 ARG142 10.97 ARG163 12.68 ARG217 13.38 ARG219 13.99 ARG303 11.91 ARG383 11.74 ARG400 11.88 ARG406 12.02 ARG420 12.60

ARG21 13.56 ARG39 11.68 ARG46 19.15 ARG62 13.23 ARG66 8.62 ARG136 13.66 ARG156 10.97 ARG162 13.44 ARG222 13.59 ARG251 12.59 ARG342 12.29 ARG365 13.25 ARG381 11.16 ARG447 12.30 /

ARG11 12.63 ARG15 17.04 ARG54 11.97 ARG56 15.00 ARG58 12.27 ARG73 12.81 ARG79 9.86 ARG127 13.28 ARG131 13.17 ARG148 12.50 ARG204 12.99 ARG225 13.45 ARG241 13.43 ARG300 13.54 ARG323 12.35

However, in the Free_HPAP, Free_HPAP_A and Free_HPAP_B models, there are only two unprotonated and neutrally charged arginines, as shown in Fig. 4. It should be attributed to the double hydrogen bonds between Asp179 and Arg15 unique in HPAP, which can effectively prevent Arg15 from losing protons. Such observations are also consistent with the results of the pKa predictions by PROPKA3.0 (Table 1). In the crystal structures of HPAP, only one of the three arginines, Arg79, gains the lowest and unusual pKa 9.86, while Arg15 gets the highest and unusual pKa 17.04. Among all these enzymes, the conserved arginine (Arg142 in ANAP, Arg156 in ANPhyt, Arg79 in HPAP) consistently has much lower pKa values. Arg136 in ANAP, Arg46 in ANPhyt, and Arg15 in HPAP, which have the highest and unusual pKa values, are all consistently paired with an aspartate residue in their crystal structures. In all these models, the arginines and the water molecules constitute a buffer mechanism for the exogenous protons (Fig. 5). Unless acidic condition is enough, the neutrally charged 11



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arginines can hardly be reprotonated, and the three water molecules (Wat1, Wat2 and Wat3) can hardly be replaced by the substrate. It is also can be concluded that the substrate in the protonated state could facilitate the substitution for water molecules, which supports the suggestion that the substrate most likely brings one proton at enzyme’s optimum pH

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summary, this mechanism, buffering protons from the acidic environment, makes sure the subsequent catalytic reaction is available in acidic conditions. More fascinatingly, there are three unprotonated arginines observed in ANAP and ANPhyt models, while there are only two in HPAP (Fig. 5). It suggests ANAP and ANPhyt can accommodate more exogenous protons and resist more acidic constraints than HPAP. It is very consistent with ANAP and ANPhyt having the much lower pH optima of 2.5 than that of 5.0 for HPAP. Since ANPhyt has another pH optima at 5.5, it can be interpreted as the phosphoryl of its special substrate phytate can still carry protons at a relatively higher pH,44,45 which facilitates the substitution for water molecules. It is known that the phytate, possessing six phosphates (each phosphate taking two protons at neutrality), loses its six second protons from pH 6.0 to 10.5,44 while the most common artificial substrate p-nitrophenylphosphate (pNPP) loses its second proton at ca. pH 5.0.46 It is dovetailed with the HPAP pH optima of 5.0 and the ANPhyt second pH optima of 5.5.

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Fig. 5. Acid proof mechanism for HAPs. Histidine nucleophilic attack To investigate the first step of the catalytic reaction of HAPs, the model was constructed on the basis of the crystal structure of HPAP (PDB code: 1nd6). Only the highly conserved active-site residues in all HAPs were considered, and the most common artificial substrate pNPP was used, whose binding mode refers to the phosphate ion in the crystal structure of HPAP (Fig. 3c). Simultaneously, WatA and WatB, observed in the crystal structure of HPAP, were added into the model successively, producing the AS-pNPP-A and AS-pNPP-B models. The AS-pNPP-A model possesses WatA, while the AS-pNPP-B model possesses both WatA and WatB. The characterizations of the reactant, transition and product states are shown in Fig. 6 and S2, and the potential energy surfaces of the reaction are given in Fig. 7.

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Fig. 6. Optimized geometries of transition states in the AS-pNPP-A and AS-pNPP-B models. The fixed atoms are marked with asterisks and the distances are in angstroms. In the AS-pNPP-A and AS-pNPP-B models, the transition states for the nucleophilic attack by the catalytic histidine on the substrate (Fig. 6) were optimized and confirmed to be the first-order saddle point with an imaginary frequency (-125.34 and -59.11 cm-1, respectively). At the transition states, the key distances between the nitrogen of the catalytic histidine and the phosphorus center of the substrate (N–P) are 2.09 and 2.00 Å respectively, while the distances between the phosphorus center and the oxygen of the leaving group 4-nitrophenyl (P–OL) are 2.62 and 2.70 Å, respectively. The calculated energetic barriers for this step are 34.8 and 32.4 kcal/mol, respectively (Fig. 7).

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Fig. 7. Potential energy profiles of the catalytic reactions of the AS-pNPP-A, AS-pNPP-B and AS-pNPP-AII models and these with CPCM solvation effects. When the polarization effects of the enzyme environments are considered, the energetic barriers drop to 21.4 and 14.8 kcal/mol for the AS-pNPP-A model with the constants ԑ=4 and 80 respectively, while they drop to 19.9 and 14.1 kcal/mol for the AS-pNPP-B model with the constants ԑ=4 and 80 respectively (Fig. 7). It would be attributed to the charged residue arginines. The polarization effect probably enhances the ability of the arginines to stabilize the negative charge of phosphate group with the arginines playing a similar role as the metal center of metallohydrolases in catalytic reactions.8,47 All the energetic barriers with the polarization effects are relatively lower than the barrier of the reaction in the study by Sharma et al. (approximately 24 kcal/mol).24 WatA has been suggested to be the likely nucleophile attacking on the phosphohistidine intermediate just because it is adjacent to the active site in the crystal structure.14,21,27 However, the optimized geometries in both AS-pNPP-A and AS-pNPP-B models showed 15



that WatA is not only held by Asp258 and Arg15 via hydrogen bonds but also connects the leaving group p-nitro-phenolate via a hydrogen bond (Fig. 6). It should not be a ready and compliant state as nucleophile for further hydrolyzing the phosphohistidine intermediate. We therefore speculated that a free water molecule from solvent would be a more appropriate nucleophile for the subsequent hydrolysis, which will be discussed in the following section. For WatB in the AS-pNPP-B model, it seems that WatB has no obvious role and makes little contribution to the energetic barrier. Phosphohistidine hydrolysis To investigate the second step of the catalytic reaction of HAPs, a water molecule named WatC, as a free water molecule from solvent, was added into the AS-pNPP-A model, producing the AS-pNPP-AII model. The optimized geometries show that WatC, at the reactant (RII in Fig. 8), forms one hydrogen bond with the oxygen of the p-nitro-phenolate and the other hydrogen bond with the atom of the phosphoryl. Thus WatC is positioned suitably as a nucleophile attacking the phosphohistidine intermediate.

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Fig. 8. Optimized geometries of reaction (RII), transition (TSII), and product (Prod) states along the reaction paths in the AS-pNPP-AII model. The fixed atoms are marked with asterisks and the distances are in angstroms. The transition state for the nucleophilic attack by WatC on the phosphohistidine intermediate (TSII in Fig. 8) was optimized and confirmed to be the first-order saddle point with an imaginary frequency -118.35 cm-1. At the transition state, the key distance between the phosphorus center of phosphoryl and the oxygen of WatC (P–OW) is 2.38 Å, while the distance between the nitrogen of the catalytic histidine and the phosphorus center (N–P) is 2.13 Å. The calculated energetic barrier for this step is 1.2 kcal/mol (Fig. 7). When the polarization effects of the enzyme environments were considered, the energetic barrier goes up to 5.7 and 8.0 kcal/mol with the constants ԑ=4 and 80, respectively, which are much lower than the barrier of the reaction in the study by Sharma et al. (18.1 kcal/mol) 26. 17



The proton transfer from WatC to the leaving group occurs simultaneously, producing p-nitrophenol at the end. Since the pKa of p-nitrophenol in the solution is 7.1,48 the leaving p-nitrophenol here can act as a general base to abstract a proton of WatC to generate a hydroxide nucleophile, and even perform better in acidic environment. In addition, the product phosphate turns out to be mono-anion by accepting a proton from Arg79 (Fig. 8). Obviously, such mono-anion phosphate is much more easily substituted by a mono-anion pNPP with one proton than a dual-anion pNPP, which is very consistent with the suggestion that the substrate most likely brings one proton at the enzyme’s optimum pH.14 In the optimization of the geometry at product state, once removing the leaving group p-nitrophenol as shown in Fig. 8, the hydrolysate phosphate will form a hydrogen bond with Asp258, which is consistent with the HPAP crystal structure as shown in Fig. 3. Therefore, the calculations, both in the structural and energetic perspectives, support our speculation that the nucleophile for attacking the phosphohistidine intermediate should be a free water molecule from solvent. Associating our observations that the active sites can accommodate three water molecules at the free stage of HAPs, the free water molecule can be a universal nucleophile for all HAPs. Then, our calculations described a complete catalytic cycle for HAPs (Fig. 9), which included the free stage before substrate binding and the catalytic reaction stage after substrate binding. At a much lower substrate concentration, the product phosphate can be substituted by water molecules, and the enzyme turns into the free stage, while at much higher substrate concentration, the product phosphate can be directly replaced by substrate. The acid condition can promote the both substitutions and increase the catalytic efficiency, which suggests a 18


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buffer mechanism for the exogenous protons in HAPs.

Fig. 9. Catalytic cycle for HAPs in acidic environments. CONCLUSION Our studies suggest a complete catalytic cycle for HAPs. This catalytic cycle reveals an acid proof mechanism for all HAP members. It also suggests a water molecule from solvent hydrolyzes the phosphohistidine intermediate to liberate phosphate and protonate the leaving group. A substantial amount of enzymatic mechanisms, suggested by theoretical studies, concentrate too much upon the chemical properties of catalytic reactions rather than the 19



biological properties of enzymes. In the present study, we suggest the enzymatic mechanisms should possess enough biological significance and satisfy two criteria. First, the proposed enzymatic mechanisms should be a logic-complete catalytic cycle. A complete catalytic cycle is the basement for understanding enzymatic functions from the mechanism perspective. Second, the proposed enzymatic mechanisms of a given enzyme should be related to its family members and the molecular environments. The enzymatic mechanisms, co-evolving with the active sites under the constraints of molecular environments, should also possess homology and adaptability, instead of coming from nothing. ACKNOWLEDGMENTS We acknowledge the National Supercomputing Center in Shenzhen for providing the computational resources and Gaussian (Gaussian 09 D01: Gaussian TCP-Linda). This work was supported by the National Natural Science Foundation of China (grant nos. 21303009, 21203042, 21573021 and 11574062), the Natural Science Foundation of Gansu Province (grant no.145RJYA285), and the Fundamental Research Funds for the Central Universities (grant no. 31920150027). SUPPORTING INFORMATION Figures of the optimized geometries, figures of potential energy profiles with the 6-311++G(2d,2p) basis set and dispersion correction, and coordinates of the optimized geometries are provided in the supporting information file. The Supporting Information is available free of charge on the ACS Publications website. REFERENCES (1)

Zalatan, J. G., Fenn, T. D. & Herschlag, D. (2008). Comparative Enzymology in the 20


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Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active-Site Metal Ion. J. Mol. Biol. 384, 1174-1189. (2)

Huang, H., Patskovsky, Y., Toro, R., Farelli, J. D., Pandya, C., Almo, S. C., Allen, K.

N. & Dunaway-Mariano, D. (2011). Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 Is an Inorganic Pyrophosphatase. Biochemistry 50, 8937-8949. (3)

López-Canut, V., Roca, M., Bertrán, J., Moliner, V. & Tuñón, I. (2011). Promiscuity

in Alkaline Phosphatase Superfamily. Unraveling Evolution through Molecular Simulations. J. Am. Chem. Soc. 133, 12050-12062. (4)

Ben-David, M., Wieczorek, G., Elias, M., Silman, I., Sussman, J. L. & Tawfik, D. S.

(2013). Catalytic Metal Ion Rearrangements Underline Promiscuity and Evolvability of a Metalloenzyme. J. Mol. Biol. 425, 1028-1038. (5)

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Table of Contents Image

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TOC 82x44mm (300 x 300 DPI)


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Fig. 1. Buffer mechanisms for PAPs against acid environments and E. coli AP against alkaline environments. H+ and OH- in red represent exogenous proton and hydroxyl ions from solution. 69x35mm (600 x 600 DPI)



Fig. 2. Reaction mechanism of HAPs for the first step (left) and the second step (right). The proton and the water molecule in red indicate their sources are undetermined. 26x8mm (600 x 600 DPI)


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Fig. 3. Active sites in the crystal structures of ANAP (PDB code: 1qfx),17 ANPhyt (PDB code: 1ihp),37 and HPAP (PDB code: 1nd6).14 The dashes show the potential hydrogen-bonds and the important distances are in angstroms. The red arrows indicate the differences in the active-site structures. These figures were created with the software Pymol.42 177x51mm (300 x 300 DPI)



Fig. 4. Optimized geometries of Free_ANAP, Free_HPAP, Free_HPAP_A and Free_HPAP_B models. The dashes show the potential hydrogen bonds and the important distances are in angstroms. The fixed atoms are marked with asterisks. 177x129mm (300 x 300 DPI)


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Fig. 5. Acid proof mechanism for HAPs. 109x85mm (600 x 600 DPI)



Fig. 6. Optimized geometries of transition states in the AS-pNPP-A and AS-pNPP-B models. The fixed atoms are marked with asterisks and the distances are in angstroms. 177x91mm (300 x 300 DPI)


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Fig. 7. Potential energy profiles of the catalytic reactions of the AS-pNPP-A, AS-pNPP-B and AS-pNPP-AII models and these with CPCM solvation effects. 93x62mm (600 x 600 DPI)



Fig. 8. Optimized geometries of reaction (RII), transition (TSII), and product (Prod) states along the reaction paths in the AS-pNPP-AII model. The fixed atoms are marked with asterisks and the distances are in angstroms. 177x115mm (300 x 300 DPI)


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Fig. 9. Catalytic cycle for HAPs in acidic environments. 143x146mm (600 x 600 DPI)


Theoretical Studies on the Catalytic Cycle of ... - ACS Publications

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