Understanding the Mechanism of Deacylation Reaction Catalyzed by

Jul 26, 2010 - Sleat , D. E.; Donnelly , R. J.; Lackland , H.; Liu , C. G.; Sohar , I.; Pullarkat , R. K.; Lobel , P. Science 1997, 277, 1802. [Crossr...
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Understanding the Mechanism of Deacylation Reaction Catalyzed by the Serine Carboxyl Peptidase Kumamolisin-As: Insights from QM/MM Free Energy Simulations Qin Xu,† Liyan Li,†,‡ and Hong Guo*,† Department of Biochemistry and Cellular and Molecular Biology, UniVersity of Tennessee, KnoxVille, Tennessee 37996, and Institute of Food and Medicine, Ocean UniVersity of China, Qingdao, 266003, P.R. China ReceiVed: March 28, 2010; ReVised Manuscript ReceiVed: July 2, 2010

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics and free energy simulations are performed to study the process of the deacylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism. The results given here suggest that Asp-164 acts as a general acid/base catalyst not only for the acylation reaction but also for the deacylation reaction. It is shown that the electrostatic oxyanion hole interactions may be less effective in transition state stabilization for the kumamolisin-As catalyzed reaction compared to the general acid/base mechanism involving the proton transfer from or to Asp-164. The dynamic substrate-assisted catalysis (DSAC) involving His at the P1 site of the substrate is found to be less important for the deacylation reaction than for the acylation reaction in the kumamolisin-As catalyzed reaction. The proton transfer processes during the enzyme-catalyzed process are examined and their role in the catalysis is discussed. Introduction Sedolisins belong to a family of enzymes (MEROPS S53) that have been identified recently and found in a wide variety of organisms.1 These enzymes normally exhibit maximum activity at low pH and often high temperature.2 It is believed that the sedolisin family might have been derived through the divergent evolution from a common ancestor with some classical serine peptidases.2 Indeed, sedolisins have a fold resembling that of subtilisin,2,3 even though they are significantly larger. Although sedolisins are still poorly understood, there is no question about their importance in biology. Indeed, a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis, is caused by the loss of the activity of the enzyme tripeptidyl-peptidase 1 (TPP1) of the family as a result of mutations in the TPP1 gene (previously named CLN2).4 Crystal structures have been available for some members of the sedolisin family,2,5-13 including kumamolisin-As to be studied in this work (Figure 1A).8,11 These structures showed that the defining features for the enzymes are a unique catalytic triad, Ser-GluAsp (Ser-278-Glu-78-Asp-82 for kumamolisin-As), as well as the presence of an aspartic acid residue (Asp-164 for kumamolisin-As) that occupies the position of Asn-155 in subtilisin (a residue that creates the oxyanion hole in the serine peptidase).14 Kumamolisin-As is a member of the sedolisin family and the first known example of a collagenase from the family. It was originally identified in the culture filtrate of a thermoacidophilic soil bacterium Alicyclobacillus sendaiensis strain NTAP-1.15 Computational studies have been undertaken to determine the similarities and differences between the catalytic mechanisms of classical serine peptidases and sedolisins for the acylation step of the enzyme-catalyzed reaction involving kumamolisin-As.16-19 One conclusion from previous quantum * To whom correspondence should be addressed. E-mail: [email protected]. Telephone: (865)974-3610. Fax: (865)974-6306. † University of Tennessee. ‡ Ocean University of China.

mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations is that the aspartic acid residue (Asp164 for kumamolisin-As) that replaces Asn155 in subtilisin may act as a general acid catalyst to protonate the substrate and stabilize the tetrahedral intermediate (TI) during the nucleophilic attack.16,17 Moreover, Asp-164 was found to act as a general base during the formation of the acyl-enzyme from the tetrahedral intermediate based on the computer simulations and may therefore play multiple roles during the catalysis. These results suggest that although sedolisins might have evolved from a common precursor with the classical serine peptidases, they seemed to have ended up with the use of different chemistry for the catalysis. It is believed that the general acid/base mechanism observed from the computer simulations on kumamolisin-As may be applied to other members of the sedolisin family as well.14 However, the enzyme-catalyzed reactions contain both acylation and deacylation steps, and the observation of the general acid/base mechanism for the stabilization of the TI for the acylation step cannot be simply extended to the deacylation step due in part to the significant changes in the active site (see below). Additional studies are necessary to establish such a general acid/ base mechanism for the catalysis by kumamolisin-As and other members of the sedolisin family. Another interesting suggestion from the previous computer simulations on the acylation step of the kumamolisin-As catalysis is that the dynamics involving the side chain of His at P1 of the substrate triggered by the bond-breaking and -making events may play an important role in the stabilization of the tetrahedral intermediate (TI1). This type of the catalytic process is normally referred to as substrate-assisted catalysis (SAC)20,21 in which certain functional group(s) from the substrate, in addition to those from the enzyme, may contribute to the rate acceleration for the enzyme-catalyzed reaction. A major difference between what was observed in the computer simulations of kumamolisin-As18,19 and many of previous studies on SAC21 is that in the former case the His residue at the P1 site is able to undergo conformational changes and form alternative hy-

10.1021/jp102785s  2010 American Chemical Society Published on Web 07/26/2010

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Figure 1. (A) Left: experimental three-dimensional structure of kumamolisin-As complexed with the inhibitor AcIPF (N-acetylisoleucylprolylphenylalaninal) obtained from ref 8. This structure was used for building the initial model for the study of the acylation reaction in refs 18 and 19, and the resulting acyl-enzyme obtained from the simulations was used for the investigation of deacylation process reported in this work. Right: activesite structure of kumamolisin-As-AcIPF complex. (B) Catalytic mechanism and the role of the key active-site residues for both acylation and deacylation reactions based on this study as well as the previous investigation (refs 18 and 19). The black arrows indicate the directions of the proton transfers and red arrows the nucleophilic attacks.

drogen bonding interactions during the acylation step. Thus, the dynamics is the key in the substrate-assisted TI stabilization in this case. This type of SAC involving conformational changes of the substrates was therefore defined as dynamic SAC (DSAC),18,19 to be distinguished from the standard SAC for

which the conformational changes of the substrate are not required for its participation in the catalysis. One advantage of DSAC is that it might lead to the stabilization of multiple reaction intermediates through the dynamics. The DSAC effect observed for kumamolisin-As could contribute to the relatively

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high specificity for the substrates with His at P1 as observed experimentally for this enzyme,8 although other factors may be involved as well. It should be pointed out, however, that although DSAC was observed for the acylation step, it does not mean that it would also exist in the deacylation step. Indeed, one of the main driving forces for DSAC in the acylation step is the salt bridge interaction between the side chains of His at P1 and Asp-164 during the formation of the tetrahedral intermediate.19 The formation of this interaction is quite sensitive to the relative position and orientation of the His and Asp-164 residues as well as their environments that may affect their flexibilities. The removal of the two Phe residues from the C-terminal region of the substrate and the existence of the covalent bond between the side chain of Ser-278 and the carboxyl group of the scissible peptide bond of the N-terminal part of the substrate in the acyl-enzyme might have a significant effect on the structure as well as the flexibility of the system that may prevent His at P1 to participate in DSAC. A detailed investigation for the deacylation step is therefore necessary for a better understanding of the role of DSAC. Here we examine the deacylation reaction catalyzed by kumamolisin-As based on the structure of the acyl-enzyme from the previous investigations16-19 using quantum mechanical/ molecular mechanical (QM/MM) molecular dynamics (MD) and free energy (potential of mean force) simulations. The results show that Asp-164 also acts as a general acid/base catalyst in the deacylation step. Moreover, the dynamic substrate assisted catalysis (DSAC) involving His at the P1 site seems to be less important for the deacylation process compared to the acylation step. The conclusions on the catalytic mechanism of kumamolisin-As determined from the previous and current simulations are summerized in Figure 1B. Methods A fast semiempirical density-functional method (SCC-DFTB)22,23 implemented in the CHARMM program24 was used in the QM/ MM molecular dynamics (MD) and free energy (potential of mean force or PMF) simulations of the deacylation process. The efficiency of the semiempirical QM methods (such as SCC-DFTB) makes it possible to sample millions of the structures and conformations for enzyme systems and to determine the free energy profiles of the enzyme-catalyzed reactions. These important tasks for understanding enzymes are not feasible with high-level first-principle ab initio methods. However, the comparisons of performance of the SCC-DFTB method on certain models as well as on simplified enzyme systems with those from high-level ab initio calculations are of importance, and such tests have been done for kumamolsin-As in our previous investigations; for more detailed discussions concerning the use of the SCC-DFTB method and comparison with high level ab initio methods for kumamolisin-As, see refs 16-19. The initial coordinates for studying the deacylation process was prepared from the structures at the end of the free energy simulation of the acylation process.16-19 In these structures, the N-terminal region of the substrate forms an acyl-enzyme with kumamolisin-As through a covalent bond between the carbonyl group of the scissile peptide bond and the side chain of Ser278. For this complex (designated as AE1), the first product from the peptide bond cleavage (the C-terminal region of the substrate) is still retained in the active site. To remove this peptide fragment, a constraint with the force constant of 100 kcal · mol-1 · Å-2 was applied to a reaction coordinate that is the average value of the two distances from the P1-His carbonyl carbon to the nitrogen of the P1′-Phe amino group and to the

Xu et al. Cβ of P1′-Phe, respectively. This reaction coordinate was gradually increased from 4.00 to 11.50 Å in 16 windows with 40 ps for each window. The resulting structure that had the first product removed from the site where the bond breaking and making takes place (designated as AE2) was then treated with two cycles of minimization (see below), heating (from 50 to 300 K in 20 ps), and equilibration (at 300 K for 80 ps). A water molecule (W1) that is close to the P1-His carbonyl carbon was changed from the MM region into the QM region (see below), and this water molecule was used later for the nucleophilic attack in the deacylation process. The system was solvated by a modified TIP3P water model25,26 using the standard procedure in the CHARMM program. The stochastic boundary MD method27 was applied to the solvated system. To be consistent with the previous study of the acylation reaction, the reaction region was the part of the system with radius R < 16 Å, and the buffer region had R equal to 16 Å e R e 18 Å. The reference center for this partitioning was chosen to be the carbonyl carbon atom (C) of the His residue at the P1 site of the substrate. The Langevin dynamics (LD) in the buffer region had frictional constants as 250 ps-1 for the protein atoms and 62 ps-1 for the water molecules. In the reaction region, the side chains of Glu78, Asp-82, Asp-164, and Ser-278, as well as a part of the substrate [the carbonyl of P2-Pro (CdO), the whole residue of P1-His, and part of P1′-Phe (CRsNH)] were treated by the quantum mechanical (SCC-DFTB) method and the rest of the system by the molecular mechanical (MM) method. The atoms in the QM region were treated with the SCC-DFTB method as described above. The all-hydrogen potential function (PARAM22)28 was used for the MM method. The link-atom approach29 available in the CHARMM program was used to separate the QM and MM regions. The resulting system contains 3430 atoms (2544 enzyme atoms, 85 substrate atoms, and 801 atoms for 267 water molecules, including 79 crystal water molecules). The same procedure was used for the study of the deacylation processes in the D164N and D164A mutants for which the acyl-enzyme structures were generated simply by replacing Asp-164 by Asn and Ala, respectively, in the AE2 complex. This allows a better comparison of the deacylation process in wild-type and mutated enzyme with the similar initial conditions. The entire stochastic boundary system was first optimized by adopted basis Newton-Rhaphson (ABNR) method. Then the system was gradually heated from 50 to 300 K in 20 ps, equilibrated at 300 K for 80 ps and then simulated at 300 K for at least 1 ns. A 1 fs time step was used for integration of the equations of motion, and the coordinates were saved every 50 fs for analyses. As discussed previously,16-19 for the acylation process the reaction coordinate is defined as R(C-N) R(C · · · Oγ), the difference between the bond length of the scissile peptide bond R(C-N) and the distance involving Oγ(Ser-278) during the nucleophilic attack. For the deacylation process, the reaction coordinate ξ is defined as ξ ) R(C-Oγ) R[C · · · O(W1)], where R[C · · · O(W1)] is the distance involving W1, which makes nucleophilic attack on P1-His carbonyl carbon. The umbrella sampling method30 implemented in the CHARMM program and weighted histogram analysis method (WHAM)31 were applied to determine the change of the free energy (potential of mean force, or PMF) for the deacylation reaction in wild-type kumamolisin-As and its mutants. In addition, the free energy profile for wild-type with the proton fixed on the carboxylate of Asp-164 using the SHAKE algorithm32 was also obtained. Sixteen ∼ nineteen windows were used to generate the free energy profiles for changing AE2 to

Acylation Reaction Catalyzed by Kumamolisin-As the product complex (EP), and for each window 100 ps simulations were performed (50 ps equilibrium and 50 ps production run). The force constants used in these free energy simulations were 100-800 kcal · mol-1 · Å-2. Results and Discussion Structure and Dynamics of the Acyl-Enzyme (AE2). The average active-site structure for the acyl-enzyme (AE2) obtained from the QM/MM MD simulations is given in Figure 2A. It is expected that W1 attacks the carbonyl carbon to form the tetrahedral intermediate. Figure 2A shows that this water molecule is well positioned for the attack during the MD simulations. Glu-78 and Asp-164 are expected to act as the general base and acid catalysts, respectively, during the attack based on previous investigations, and the active-site structure is consistent with the expected role of these residues. Asp-82 forms a low-barrier hydrogen bond with Glu-78 in AE2 (middle panel on the right-hand side of Figure 2A); a similar low-barrier hydrogen bond was found to be formed in AE1 as well.19 Figure 2A (top panel) shows that Asp-164 interacts with the carbonyl group of the P1-His with Ha mainly located on Asp-164. This is consistent with the expectation that the role of this residue is to stabilize the tetrahedral intermediate either through the general acid/base mechanism (i.e., similar to its role in the acylation step of the kumamolisin-As catalyzed reaction) or by the strengthening of the interaction during the charge formation (i.e., as in the case of classical serine peptidases); the exact role of Asp-164 for deacylation will be discussed later. The hydrogen bonding interactions from the backbone amide group of Thr277 as well as its side chain seem to be crucial in stabilizing the position of Asp-164. Given the important function of Asp164, such stabilization should be of importance. In addition to Asp-164, the amide group of Ser-278 also interacts with the carbonyl oxygen (i.e., similar to the case of classical serine peptidases). The P1-His side chain forms a stable hydrogen bond with the carbonyl group of P2-Pro, and this is the residue that has been suggested to play a role in dynamic substrateassisted catalysis during the acylation reaction.18,19 Comparison of the Free Energy Profiles of the Acylation and Deacylation. Figure 2B compares the changes of free energy (potential of mean force) profiles as a function of the reaction coordinate (ξ) for the deacylation reaction (right) with those of the corresponding acylation reaction (left). In addition to free energy profiles for wild-type, D164N and D164A, the free energy profiles for the case in which the proton on Asp164 is fixed by the Shake algorithm32 are also given (to prevent Asp-164 acting as the general acid/base catalyst for the nucleophilic attacks and the formation of the acyl-enzyme or product). The curves of the free energy profiles for the deacylation reaction are shifted such that the relative stabilities for AE1 and AE2 are similar. Although this may make it easier for comparison, it should keep in mind that AE1 and AE2 are in different stages of the enzyme-catalyzed reaction and are not the same: i.e., AE1 has the cleaved C-terminal part of the substrate remained at the site where the acylation reaction took place, while AE2 does not have it. Thus, the free energy profiles do not provide any information as to whether the acylation or deacylation process is rating limiting. It should be pointed out the free energy profiles in Figure 2B were based on the onedimensional free energy simulations, as the determination of the multidimensional free energy profiles is very time-consuming. For the stepwise bond breaking and making events, the use of one-dimensional free energy simulations may not pose a serious problem. However, for the concerted processes some

J. Phys. Chem. B, Vol. 114, No. 32, 2010 10597 information that would be contained in the multidimensional free energy profiles may be absent in the one-dimensional profiles. Indeed, there are several proton transfers during the acylation and deacylation reactions, and some of these proton transfers may be coupled to the rearrangement of the system (i.e., the nucleophilic attacks and the formation of the acylenzyme or product). For instance, our earlier two-dimensional free energy simulations on the nucleophilic attack of Ser-278 on the AcIPF inhibitor in kumamolisin-As showed that there are important free energy relationships between the nucleophilic attack and the proton transfers. Such free energy relationships between the nucleophilic attack and proton transfers are absent in the one-dimensional free energy profiles (Figure 2B). However, the previous study17 also indicated that onedimensional free energy simulations with the selection of the suitable reaction coordinate (as we did here) should be sufficient to determine the key energetic properties for the acylation and deacylation reactions in kumamolisin-As. Figure 2B (right) shows that TI2 is considerably more stable in wild-type than in D164N, leading to the relatively low free energy barriers for the TI2 and EP formation. This result suggests that for the deacylation process in kumamolisin-As the general acid/base mechanism is more effective in lowering the energy barriers than the stabilization of the charge formation on the carbonyl oxygen through the hydrogen bonding interaction (i.e., by Asn-164 in the D164N mutant), consistent with the results of the previous investigations of the acylation process (Figure 2B, left).18,19 It is of interest to note that the barrier lowering in going from D164N to wild-type for deacylation is somewhat smaller than that for acylation (see below). Although the barriers for the formation of TI2 and EP are higher for D164N compared to those for wild-type, they are still considerably lower than the barriers for D164A. This indicates that the hydrogen bonding interactions from Asn-164 can make a very significant contribution to the transition state stabilization, although other factors, such as the cooperative effect of hydrogen bonding33-35 from Thr-277, may be involved as well. The free energy profile of D164N is quite similar to the one for which the proton on Asp-164 is fixed by the Shake algorithm, confirming the suggestion that the barrier lowering is due to the proton transfer. Comparison of the free energy profiles for the acylation and deacylation steps in Figure 2B shows that one major difference for these two processes is that while TI1 seems to be a stable conformation only in wild-type, TI2 is stable in the both wild-type and mutants. The results of the simulations reported here extend the earlier proposal16,17 for the importance of the general acid/base mechanism involving Asp-164 to the stabilization of TI2 in the deacylation process and are consistent with the available mutagenesis studies. For instance, the D164A mutant of kumamolisin did not show any measurable proteolytic activity.12 For the D164N mutant of kumamolisin-As, a low but appreciable level of catalytic activity (1.3%) was found,11 corresponding to an increase of the activation barrier of about 2.5 kcal/mol. This increase of the activation barrier seems to be similar to the increase of the free energy barrier for the deacylation process. However, it should be pointed out that care must be exercised in making direct comparisons of the computational results with experimental data. For instance, it is not clear whether the acylation or deacylation reaction is ratelimiting. Moreover, the processes of the substrate binding and product release need to be considered as well. Furthermore, structural analyses of the unliganded D164N mutant showed that, although the catalytic triad is intact, the mutated residue (Asn-164) unexpectedly makes a hydrogen bond with the side

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Figure 2. (A) Left: average structure of the active site of the acyl-enzyme that was formed after the acylation step of the reaction involving the GPH*FF substrate (refs 18 and 19) as well as release of the cleaved C-terminal region of the substrate from the active site (see text). The acyl-enzyme is termed as AE2 to distinguish it from the one (AE1) in which the C-terminal region of the substrate still remains at the active site after the cleavage (ref 19). Most of the hydrogen atoms are not shown here for clarity. Right: distances (Å) from certain oxygen atoms to three hydrogen atoms, Ha, Hb, and Hc, as functions of time (ps). These atoms may be involved in proton transfer processes during the deacylation step (see below). Top panel: the distances from O (D164) and O (carbonyl group of P1-His) to Ha (colored in blue and magenta, respectively). Middle panel: the distances from O (E78) and O (D82) to Hb (colored in blue and magenta, respectively). Bottom panel: the distances from O (W1), O (E78), and O (S278) to Hc (colored in blue, magenta, and green, respectively). The locations of the hydrogen atoms, Ha, Hb, and Hc, are shown in the structure on the left. (B) Left: free energy (potential of mean force) profiles for the acylation reaction from ref 19. For the acylation reaction, the reaction coordinate is ξ ) R(C-N) - R(C-Oγ) (ref 19). Right: free energy profiles for the deacylation reaction of this work. The reaction coordinate for deacylation is ξ ) R(C-Oγ) - R(C-O); see Figure 2A for the definition of the atoms. The curves of the free energy profiles for the deacylation reaction are shifted such that the relative stabilities for AE1 and AE2 are similar. This may make it easier for comparison. TI1 and TI2 are the tetrahedral intermediates for acylation and deacylation, respectively. TSa1 (TSa2) and TSd1 (TSd2) are the first (second) transition state for acylation and deacylation, respectively. Key: wild-type kumamolisin-As, red and solid; D164N, green and dot; D164A, magenta and dash. In addition, the free energy profile was also obtained for the wild-type enzyme with the proton fixed on D164 with the SHAKE algorithm to prevent the proton transfer (blue and dot-dashed line).

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Figure 3. The active-site structures and the distances to the hydrogen atoms (protons) as functions of time in the first transition state TSd1 of the deacylation step (A), the tetrahedral intermediate TI2 (B), the second transition state TSd2 (C), and the product complex (D). Top panel: distances from O (D164) and O (carbonyl group of P1-His) to Ha colored in blue and magenta, respectively. Middle panel: distances from O (E78) and O (D82) to Hb colored in blue and magenta, respectively. Bottom panel: distances from O (W1), O (E78), and O (S278) to Hc colored in blue, magenta and green, respectively.

chain of Ser-278.11 The consequence of this structural change and the way by which the side chain of Asn164 stabilizes the transition state in the reaction catalyzed by the D164N mutant are still unknown. Structures of TSd1, TI2, TSd2, and EP and Proton Transfers. The average active-site structures of TSd1 (near the transition state for the nucleophilic attack by W1), TI2 (the tetrahedral intermediate of deacylation), TSd2 (near the transition state for the formation of the product complex), and EP (product complex) are given in Figure 3 along with the fluctuations of some distances involved in the proton transfers that are coupled with the deacylation reaction. The average structure in Figure 3A shows that a low-barrier hydrogen bond is formed between Asp-164 and the carbonyl group of P1-His, and this is supported by the fluctuations of the distances of Ha to O (Asp-164) and O (carbonyl group of P1-His), respectively, in the top panel on the right. Moreover, the proton on W1 (Hc) has already moved to Glu-78 at TSd1, whereas Hb is now mainly located on Asp82 (due to the protonation of Glu-78). Kumamolisin-As was found to have a higher specificity for the substrates with a positively charged residue (such as His or Arg) at the P1 site than for those they do not.8,36 It was found

in our earlier simulations18,19 that the side chain of His at P1 rotated significantly during the transition from the substrate complex to the tetrahedral intermediate (TI1) in the acylation step. This movement was triggered by the deprotonation/ protonation events and resulted in the interaction of His at P1 with the unprotonated Asp-164. It was suggested that the dynamics involving the His residue at the P1 site might play an important role in the catalysis and contribute to the higher specificity for the substrates with His at P1. Figure 3B shows that for TI2 P1-His mainly interacts with the carbonyl oxygen of P2-Pro (as observed in the acyl-enzyme AE2; see Figure 2A). This is in contract with the case of the acylation reaction.18,19 We have observed during the simulations that P1-His underwent a conformational change to interact with Asp164 from time to time in TI2. But it was far less common than in the case of the acylation process. This suggests that DSAC seems to play a less important role for deacylation than for acylation. Consistent with this suggestion, the barrier lowering for the deacylation process is less significant compared to that for acylation process (Figure 2B). The locations of the three protons (Ha, Hb, and Hc) at different stages of the reaction in wild-type are plotted in Figure 4. Figure

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Figure 4. Locations and transfers of the protons among AE2 (green), TI2 (blue), and EP (magenta) in wild-type. (A) Ha is located on D164 in both AE2 (green) and EP (magenta) states with the H · · · O (D164) distance at about 1 Å. However, this proton moves to the carbonyl oxygen of the His residue at P1 of the substrate when the tetrahedral intermediate (TI2) is formed (blue). (B) While Hb spends most of the time on D82 in TI2 (blue), it is involved in a low-barrier hydrogen bond in AE2 (green) and EP (magenta). (C) Hc moves to E78 as a result of the formation of TI2 (from green to blue) and then moves to S278 (magenta) during the formation of the product complex.

4A shows that Ha is located on Asp-164 in both AE2 (green) and EP (magenta) states with the Ha-O (Asp-164) distance at about 1 Å. However, this proton moves to the carbonyl oxygen of the His residue at P1 of the substrate when the tetrahedral intermediate (TI2) is formed (blue). Figure 4B shows that while Hb is mainly located on Asp-82 in TI2 (blue), it is involved in a low-barrier hydrogen bond in AE2 and EP and spends a lot of time in the middle of the Asp-82 and Glu-78 residues. In Figure 4C, Hc moves to Glu-78 as a result of the formation of TI2 (from green to blue) and then moves to Ser-278 (magenta) during the formation of the product. Conclusions The deacylation reaction catalyzed by kumamolisin-As has been studied by the QM/MM MD and free energy simulations. The results given here suggested that Asp-164 acts as a general acid/base catalyst not only in the acylation step but also in the deacylation step. The role of dynamic substrate assisted catalysis (DSAC) involving His at the P1 site was also discussed, and it was suggested that DSAC may be less important for the deacylation process than for the acylation process. Acknowledgment. We thank Drs. Alex Wlodawer, Toru Nakayama, and Haobo Guo for useful discussions and Professor Martin Karplus for a gift of the CHARMM program. This work was supported by National Science Foundation (Grant number: 0817940 to H.G.). References and Notes (1) Siezen, R. J.; Renckens, B.; Boekhorst, J. Proteins: Struct. Funct. Bioinf. 2007, 67, 681.

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