Mechanistic Pathway on Human Î±-Glucosidase Maltase

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B: Biophysical Chemistry and Biomolecules

Mechanistic Pathway on Human #-Glucosidase MaltaseGlucoamylase Unveiled by QM/MM Calculations Natércia Fernandes Brás, Diogo Santos-Martins, Pedro A. Fernandes, and Maria João Ramos J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01321 • Publication Date (Web): 16 Mar 2018 Downloaded from http://pubs.acs.org on March 17, 2018

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The Journal of Physical Chemistry

Mechanistic Pathway on Human α-Glucosidase Maltase-Glucoamylase Unveiled by QM/MM Calculations

Natércia F. Brás*, Diogo Santos-Martins, Pedro A. Fernandes and Maria J. Ramos

REQUIMTE/UCIBIO, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal

*correspondence should be addressed to Natércia F. Brás ([email protected])

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Abstract The excessive consumption of starch in human diets is associated with highly prevalent chronic metabolic diseases such as type 2 diabetes and obesity. α-glucosidase enzymes contribute for the digestion of starch into glucose and are thus attractive therapeutic targets for diabetes. Given that the active sites of the various families of α-glucosidases have different sizes and structural features, atomistic descriptions of the catalytic mechanisms of these enzymes can support the development of potent and selective new inhibitors. Maltase-glucoamylase (MGAM), in particular, has a N-terminal catalytic domain (NtMGAM) that has shown high inhibitor selectivity. We provide here the first theoretical study of the human NtMGAM catalytic domain, employing a hybrid QM/MM approach with the ONIOM method to disclose the full atomistic details of the reactions promoted by this domain. We observed that the catalytic activity follows the classical Koshland double-displacement mechanistic pathway that uses general acid and base catalysts. A covalent glycosyl-enzyme intermediate was formed and hydrolyzed in the first and second mechanistic steps, respectively, through oxocarbenium ion-like transition state structures. The overall reaction is of dissociative type. Both transition state geometries differ from those known to occur in other glycosidases. The activation free energy for the glycosylation rate-limiting step agrees with the experimental barrier of 15.8 kcal.mol-1. Both individual mechanistic steps of the reaction are exoergonic. These structural results may serve as basis for the design of transition state analogue inhibitors that specifically target the intestinal NtMGAM catalytic domain, thus delaying the production of glucose in diabetic and obese patients.


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Introduction Starch is one of the most used carbohydrate sources in human diet. However, its excessive consumption may result in chronic metabolic diseases, such as type 2 diabetes and obesity, due to the presence of high glucose levels in blood1. The human digestive system has four different αglucosidases that work in strong synergy to allow for an efficient and complete digestion of starch23

. First, the salivary and pancreatic α-amylases endohydrolases cleave the internal α-1,4 glycosidic

bonds of starch into maltose, maltotriose, and other α-1,6 and α-1,4 oligoglucans4. Subsequently, these shorter saccharides are hydrolyzed into glucose units in the small intestinal lumen by the exohydrolases, maltase-glucoamylase (MGAM) and sucrose-isomaltase (SIso)5. Both MGAM and SIso enzymes are bound to the intestinal membrane, and they are composed by the N-terminal (NtMGAM and NtSIso) and C-terminal (CtMGAM and CtSIso) independent catalytic domains6-7. These four catalytic subunits display overlapping substrate specificities, in particular for substrates with α-1,4 glycosidic linkages. However, this catalytic redundancy is necessary due to the 19:1 distribution ratio of α-1,4 and α-1,6 glycosidic bonds present in starch8. Both MGAM and SIso belong to glycoside hydrolase family 31 (GH31 from CAZY classification) and work through a retaining mechanism, in which the configuration of the anomeric carbon (C1) is maintained6, 9. As proposed by Koshland in 1953,10 the retaining GHs catalytic mechanism (illustrated in Scheme 1) uses both a general acid and general base catalysis and occurs via a double displacement mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. Previous experimental11 and theoretical studies12-13 provided that each step is characterized by an oxocarbenium ion-like transition state (TS). These geometries have a dissociative character that is related with the positive charge accumulated on the saccharide ring. At the TS (or nearby), the lone-pair electrons of the ring oxygen delocalize and causes a structural distortion of the pyranose ring from its lowest energy conformation14-16. In addition, some


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theoretical calculations revealed that distinct glycosidases have TS with different extension of dissociative character. α-mannosidase II17, lysozyme18, β-endonuclease19, cellulase Cel7A20, βhexosaminidase A21 and α-amylase22 exhibit transition states that are much more dissociative in nature than cellulase Cel5A23, O-glycoprotein 2-acetamino-2-deoxy-β-D-glucopyranosidase (OGlcNAcase)24-25, β-galactosidase26, cellobiohydrolase I (CBHI)27, 1-3,1-4-β-glucanase28, αgalactosidase29 and N-acetylglucosaminidase30.

Scheme 1 – Schematic representation of the reversible reaction catalyzed by both domains of the human MGAM and SIso enzymes. The NtMGAM and NtSIso domains share ca. 60% of their amino acid sequence, and 40% of sequence identity with each respective Ct domain6, 31. The MGAM shows higher enzymatic activity than SIso2, 8. Previous studies revealed that both subunits of MGAM possess independent catalytic


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activities8. Although, both NtMGAM and CtMGAM catalyze the same reaction, hydrolyzing linear α-1,4-linked oligosaccharide substrates, they have different substrate specificities. In particular, their different specificities are related to the size of the substrates7. The active site of NtMGAM is composed of a shallow substrate-binding pocket with only two sugar subsites (-1 and +1 subsites), and it preferentially cleaves disaccharides with a high prevalence for maltose5. However, the presence of an additional 21 residues in the active site of CtMGAM allows for two additional sugar subsites, which favors the binding of longer substrates8,

32

. Recently, it was found that the

NtMGAM subunit can also hydrolyze branched α-1,6-linked sugars such as isomaltose, whereas with lower efficiency33. The NtMGAM subunit is ca.100 kDa and consists of 868 residues disposed into five structural domains. When the substrate binds to the active site, its non-reducing end interacts with the buried subsite, leaving the reducing end directed to the surface exposed subsite. The active site pocket of apo-NtMGAM and NtMGAM–acarbose structures does not show major structural differences7 (see Figure S1 in Supporting Information (SI)). From a therapeutic point of view, the understanding of these enzymatic catalytic mechanisms is crucial because the use of α-glucosidase inhibitors has been proven to be efficient in delaying the glucose production, which then further regulates postprandial blood glucose levels and reduces the damage and complications in diabetic and obese patients34-35. Currently, acarbose (glucobay®, precose®), miglitol (glyset®), voglibose (basen®) and 1-deoxynojirimycin (DNJ) (duvoglustat®) are the commercially-available reversible α-glucosidase inhibitors with carbohydrate scaffold structures32, 36. The protonated forms of these nitrogen-carbohydrate molecules act by mimicking the shape and charge of the oxacarbenium ion-like transition state (TS) of the α-glucosidase catalytic mechanisms. Although these drugs have been used for two decades, they still have several side effects and complications1, 36. NtMGAM and CtMGAM also show different sensitivities to


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inhibitors due to their different number of sugar subsites and some structural differences in their active sites. For example, contrarily to CtMGAM, the NtMGAM subunit is poorly inhibited by the tetrasaccharide acarbose because its active site lacks the additional +2 and +3 sugar subsites7. As a result, the design of inhibitors with high selectivity towards a specific intestinal α-glycosidase is a huge gap in the field. For this reason, and despite the already described mechanistic studies of other glycoside hydrolases22,

26, 37

, it is important to unveil the specificities of the NtMGAM catalytic

mechanism with atomistic details, which are still not known. In the present study, we seek to elucidate with computational multi-resolution hybrid approaches the complete glycosidic bond cleavage reaction promoted by NtMGAM. The transition state geometries obtained here will be very useful to propose novel TS analogue inhibitors that can work as an alternative to the currently employed drugs that show harmful side effects.

Methods Molecular Modelling. The X-ray structure of the N-terminal domain of the human maltaseglucoamylase (NtMGAM) bound to acarbose (2QMJ.pdb, at 1.9 Å resolution)7 was used in the present study. The missing residue (Q837) was modelled from the 3L4U.pdb structure (human NtMGAM crystalized with kotalanol)36. The pKa values of all ionizable residues were evaluated using the PROPKA program38, and hydrogen atoms were added according to this evaluation. All residues were at their physiological protonation state with the exception of His831 (Glu862), Asp261 (Leu259), Asp336 (Phe337, Ile369), Asp366 (Val365), Asp542 (catalytic proton-donor residue) and Glu559 (Phe562), Glu788 (Glu767, Leu786), which have high pKa values (> 7.0) due to the presence of neighbor residues with hydrophobic and/or negatively charged character (indicated in parenthesis). The substrate maltose was modelled from the acarbose molecule. Twenty-three sodium counter-ions were added to neutralize the system. Explicit solvent (TIP3P water model) was used, filling a rectangular box with a 12 Å distance to any atom of the complex. The NtMGAM:maltose geometry was optimized in two steps: 1) only the solvent and counter-ions


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were minimized (500 steps using the steepest-descent algorithm (step-desc alg) and 3000 steps using conjugate gradient (conj grad)); and 2) entire system was optimized (3000 steps using the step-desc alg and 12000 steps using conj grad). The AMBER 12.0 software package39 was used to perform the optimizations. The protein and maltose were described with the ff99SB40 and Glycam0641 force fields, respectively. Molecular Dynamics (MD) simulation. Starting from the optimized geometry, 100 ps of an MD simulation using a NVT ensemble and periodic boundary conditions was performed. Further, 500 ps (with the position of protein and substrate restrained with a constant force of 20 kcal.mol-1Å-2) and 10 ns of MD simulations using an isobaric-isothermal ensemble were carried out. The temperature and pressure were maintained at 310.15 K and 1 atm using the Langevin dynamics (1.0 ps-1 as collision frequency) and the Berendsen Barostat, respectively. The PMEMD tool of the AMBER 12.0 simulation package was used to carry out all simulations. The SHAKE and the Verlet leapfrog algorithms were used to constrain the bond lengths with H atoms and to integrate the equations of motion (time-step of 2 fs), respectively. The long-range interactions were considered by the Particle-Mesh Ewald method, and the non-bonded interactions were truncated with a 10 Å cutoff. QM/MM calculations. The optimized NtMGAM:maltose structure was used as starting geometry for the QM/MM calculations. Considering the size of the complete NtMGAM:maltose complex (ca. 20,000 atoms (13,600 atoms without solvent)), we worked with a reduced model comprising a 25 Å radius sphere cut around the maltose molecule. The final system was composed by 6,695 atoms (including some water molecules). The ONIOM formalism42 employed in Gaussian 09 software43 was used to explore the potential energy surface (PES) of the NtMGAM-catalyzed reaction. For that, two layers were defined. An high-level layer that includes the entire substrate and side chains of Tyr214, Asp327, Asp366, Trp406, Asp443 (catalytic nucleophilic residue), Met444, Arg526, Trp539, and Asp542 (catalytic proton-donor residue) making a total of 142 QM atoms. A list of these atoms was included in Table S1 in SI. The density functional theory (DFT), in particular the


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B3LYP/6-31G(d), level of theory was used to describe the high-layer for geometry optimizations. It provided accurate results in various enzymatic mechanistic studies44-46. The remaining atoms of the system were described at the molecular mechanics (MM) level of theory (ff99SB and Glycam-06 force fields). The outer 5 Å shell of atoms of the entire system was frozen, to avoid the denaturation of the outer regions of the enzyme due to the spherical cut that was introduced. The mechanical electronic embedding scheme was used to describe the Coulomb interaction between the two layers. The mechanical embedding scheme is widely used, and it has successfully unveiled the reaction mechanism of various enzymes, such as renin47, isomerase PhzF48, β-galactosidase26 and the ornithine cyclodeaminase49, regardless of its associative/dissociative character. Nevertheless, special attention should be paid when defining the high layer, in particular for charge-transfer processes, to properly describe the main chemical interactions taking place in the active site; because only an accurate description of the electronic environment of the reaction can minimize the absence of polarization of the QM wave function by the MM environment system along the PES. For both mechanistic steps, linear transit scans along first putative reactions coordinates (distance between the anomeric carbon atom (C1) and the glycosidic oxygen or water oxygen atom, as well as distance between the proton of the catalytic proton-donor residue and the glycosidic oxygen or water oxygen atom) were performed. The energy maximum obtained from the PES along each reaction path corresponds to a first guess of the transition state (TS) geometry. We have used 0.05 Å increments for the reaction coordinate scan that were decreased to 0.01 Å increments when near the TS. Subsequently, the guessed TS structures were freely optimized to a TS, and the presence of a unique imaginary frequency was confirmed by the calculation of the vibrational frequencies. The zero-point energy as well as the entropic and thermal contributions to the free energy were determined at the same level of theory, within the rigid rotor/harmonic oscillator formalism and the combined free-rotor/harmonic oscillator approximation for a threshold vibrational temperature of 120 K (~100 cm-1) as proposed by Grimme et al50. All free atoms (3357 atoms) of the system were


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considered in the Hessian evaluation. Then, we moved the reaction coordinate slightly towards the reactants and products of each mechanistic step, and these structures were freely optimized, falling into the reactants and products, and the vibrational frequencies were also calculated. The geometries of all stationary points are included in the Supporting Information (SI). The barriers and the reaction free energies along the mechanism were determined using the geometries of all stationary points. The energies of these optimized geometries were obtained by Single-point (SP) calculations using different density functionals (B1B95, MPW1B95, CAM-B3LYP, PBE1PBE, wB97X-D and BB1K) and a more complete basis set – 6-311+G(2d,2p). We specifically chose these density functionals because they generally give a good agreement in activation and reaction energies for this kind of reactions21-22,

26

. Additionally, a recent benchmarking

study for the reaction catalyzed by

glycosidases indicates that the first five density functionals present errors below 0.5 kcal.mol-1, in relation to the reference energies calculated at the CCSD(T)/CBS//MP2/aug-cc-pVTZ level of theory.51 The inclusion of dispersion effects was not evaluated because previous work concluded that the D3 correction had a small influence in the geometries of the stationary points glycosidic cleavage reactions (Mean Unsigned Error (MUE) of 0.01 Å between the B3LYP and B3LYP-D3 density functionals), as well as it did not affect significantly the kinetics and thermodynamics of this reaction.51 However, in order to validate the starting geometry of our model, we have optimized our reactant state with the B3LYP-D3 method to describe the QM part (Figure S2 in SI). Visually, both optimized structures seem quite similar. The RMSd value of the QM region is 0.148 Å, while the MUE of the main distances and dihedral angle (described in Table 1) are 0.08 Å (which is a small value taking into account the non-covalent character of the mostly bonds considered) and 3.01º, respectively. In light of these results, B3LYP seems to be an adequate density functional to study the structural features of this enzyme reaction.

Results and Discussion Considering that the non-catalyzed reaction to break glycosidic linkages has a half-life of about 5


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million years13, glycosyl hydrolases such as the human NtMGAM, play a key role in this process, because they have kcat values of up to 1000 s-1.52 In the present work, we studied the catalytic mechanism by which NtMGAM cleaves the maltose molecule with net retention of anomeric configuration. Figure 1 shows our QM/MM model of the NtMGAM domain, where we also highlight the atoms included in the QM region.

Figure 1. Representation of the QM/MM model of the NtMGAM domain. A zoom of the QM region is also shown. Only the polar hydrogen atoms are depicted. Truncation sites for the QM region are marked with an asterisk. As seen in Fig. 1, the active site of NtMGAM is exposed to the solvent, and the two glucose rings (gluc-1 and gluc-2) of maltose properly occupy the -1 and +1 sugar subsites of its active pocket. As the first glucose unit of the disaccharide was modelled from the valienamine ring of acarbose, it already adopts a distorted 2H3 half-chair sugar conformation36. Numerous hydrogen bonds (Hbonds) involving the hydroxyl groups of the sugar rings and the Asp327, Asp443, Arg526, Asp542, and His600 residues, contribute to this geometry. The presence of bulky residues at the -1 sugar subsite, such as Trp406 and Trp559, can likely contribute to stacking interactions that properly orient the maltose rings. All these residues are highly conserved among the GH31 family12, 36, while some of them were indicated as relevant for the tight binding of α-glucosidase inhibitors36. Asp443


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was identified by mutagenesis studies as the catalytic nucleophile residue6. Considering that Asp542 is ca. 6 Å away from Asp443, it was pointed out as a good candidate for the acid/base catalyst7. Furthermore, Asp366 forms a water-mediated H-bond with Asp443, while Tyr214 has a hydrogen bridge with Asp542, consistent with the idea that they may play a role in stabilizing and modulating the pKa values of the two catalytic residues throughout the glycosylation step of the reaction. It is important to highlight that the H-bonds and hydrophobic interactions established in the X-ray structure were preserved in the optimized geometry (displayed in Figure 2a). However, to evaluate the stability and position of the substrate into the NtMGAM binding pocket, a MD simulation (10 ns) was performed. The analysis of the MD trajectories revealed that it remained in the same site throughout the entire simulation. Figure S3 in SI illustrates the root-mean-square deviation (RMSd) values for the enzyme backbone elements, QM region and maltose molecule. These small values (average of 0.95 ± 0.08 Å, 0.98 ± 0.13 Å and 0.96 ± 0.20 Å, respectively) support the high stability of the optimized NtMGAM:maltase complex. In fact, the most frequent H-bonds (between enzymesubstrate) during the MD simulation (see Table S2 in SI) were those observed in the optimized geometry. Glycosylation step We obtained a Potential Energy Profile (PEP) for the first mechanistic step (called glycosylation step) along the elongation of the glycosidic bond length. The distorted conformation of gluc-1 places the anomeric carbon (C1) and the glycosidic oxygen (O1) atoms in adequate positions to favor the nucleophilic attack and acid/basis catalysis by Asp443 and Asp542 residues, respectively. Several studies also suggested that the sugar ring distortions help to accelerate the rate of the reaction15-16. In particular, for the retaining GH31 α-glycosidase family it was proposed that the gluc-1 ring proceeds by a 4C1 → 4H3 → 1S3 sugar conformational change, from the substrate binding that evolves to the TS conformation, and subsequent covalent glycosyl-enzyme intermediate


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formation12. Table 1 lists the main distances, the C5-O5-C1-C2 dihedral angle and the atomic charges of relevant atoms along the reaction pathway. The transition state geometry for the first mechanistic step (TS1) is displayed in Figure 2b, in which we can see that the glycosidic bond is completely broken (2.83 Å) and the proton from the acidic Asp542 is already bound to the glycosidic oxygen (1.00 Å). The nucleophilic Asp443 residue comes closer to the C1 atom, with one of its oxygens attacking it, whereas the respective covalent bond is only established in the products of this mechanistic step. The distances between the C1 and O-Asp443 atoms are 3.26 Å, 2.05 Å and 1.49 Å in the reactants, TS1, and intermediate (INT1), respectively. Thus, at the end of the glycosylation step, a covalent glycosyl-NtMGAM intermediate is formed (see Figure 2c). From the very loose TS1 geometry, it is clear that the leaving group dissociates more quickly than the nucleophile associates to form a bond, as well as indicate that the proton transfer from Asp542 to the O1 is prior to the nucleophilic attack by O-Asp443 to the C1. Thus, the reaction is of dissociative type, as is generally accepted for glycosidases.

Table 1. Main distances, the C5-O5-C1-C2 dihedral angle and the atomic charges of relevant atoms along the reaction pathway.

bond (Å)

R

TS1

INT1

INT2

TS2

P

INT2'

TS2'

P'

C1 - O1 / Owat

1.45

2.83

3.40

4.01

1.68

1.47

3.89

2.12

1.45

C1 - Onuc OAsp542 - HAsp542 / Owat Hwat

3.26

2.05

1.49

1.50

2.96

3.13

1.51

2.80

3.13

1.02

1.65

1.77

0.97

1.09

1.69

0.99

1.03

1.74

HAsp542 - O1 / Hwat - OAsp542

2.57

1.00

0.99

2.52

1.40

1.00

1.86

1.51

1.00

C1 - O5

1.39

1.29

1.39

1.38

1.34

1.38

1.37

1.28

1.29

H2 - Onuc

1.73

1.81

3.99

3.97

4.57

4.55

2.51

1.76

1.81

H2 - OAsp542

3.42

4.10

1.83

1.72

1.85

2.67

3.54

4.32

4.10

OAsp542 - HOTyr214

2.39

1.79

1.87

1.70

1.81

2.16

1.63

1.67

1.94

-44.68

-7.81

6.75

19.37

-30.75

-44.85

16.41

-23.46

-47.01

dihedral (º) C5-O5-C1-C2 charges (a.u.)


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C1

0.22

0.31

0.35

0.31

0.34

0.30

0.33

0.34

0.47

O1 / Owat

-0.53

-0.69

-0.67

-0.84

-0.72

-0.70

-0.87

-0.83

-0.26

O5

-0.51

-0.40

-0.54

-0.52

-0.46

-0.50

-0.52

-0.46

-0.50

O2

-0.75

-0.69

-0.69

-0.69

-0.67

-0.66

-0.68

-0.67

-0.27

Onuc

-0.59

-0.57

-0.47

-0.50

-0.62

-0.61

-0.50

-0.57

-0.58

OAsp542

-0.09

-0.57

-0.60

-0.60

-0.65

-0.13

-0.55

-0.57

-0.57

Figure 2. Representation of the reactant (R), transition state (TS1) and the glycosyl-NtMGAM intermediate (INT1) geometries obtained for the glycosylation step. The main interactions established are highlighted. Only the polar hydrogen atoms are depicted.


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We also verified a distortion of the pyranose ring of gluc-1 on the TS geometry towards the distorted 4H3 half-chair sugar conformation that is typical of the oxocarbenium ion character transition states. This distortion is a consequence of the decrease in C1-O5 bond length (1.39 Å and 1.29 Å at the R and TS1 states, respectively) along with the planarity of C5, O5, C1, and C2 atoms (dihedral angle changes from -44.68° at the reactant to near planar (-7.81°) at the TS1), favoring the sp2-like hybridization and positive charge at the C1. The atomic charge of the O5 and O1 atoms increases and decreases in approximately 0.1 a.u., respectively. In addition, to stabilize the electrondeficit of C1 by orbital overlapping and electron delocalization of the ring oxygen’s lone-pair, the ring distortion minimizes steric hindrance around C1, assisting the nucleophilic attack by Asp443. Figure S4a in SI shows the superimposition of the sugar ring conformations in the three stationary points of this mechanistic step. These results are quite similar to the calculations performed on various GH enzymes. The largest differences are the extension of the dissociative character of the TS and the position of the proton from the acid/base group at the TS. The TS1 of NtMGAM has a much smaller C1-Onuc distance than C1-O1 distance (difference of 0.76 Å). High differences were also observed in the reactions of α-mannosidase II17, lysozyme18, β-endonuclease19, cellulase Cel7A20 and β-hexosaminidase A21. However, the TS1 of NtMGAM is much more dissociative in nature than the TS observed in the pancreatic α-amylase22, β-galactosidase26 and cellobiohydrolase I (CBHI)27 reactions. We note that not only enzymes that cleave different substrates can have varied dissociative TS geometries, but enzymes that hydrolyze the same substrate exhibit different TS structures. This means that the nature of the saccharide units and the type of cleaved glycosidic bond are not the sole responsible for the different dissociative TS geometries. Upon comparison of the active sites and mechanisms of the α-mannosidase II, NtMGAM, α-amylase, β-galactosidase and CBHI enzymes, we hypothesized that the presence and proximity of a positively charged residue/ion to the Onuc atom


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is directly related with the extension of the dissociative character of TS (α-mannosidase II > NtMGAM > α-amylase > β-galactosidase > CBHI). In fact, the α-mannosidase II has a zinc cation that directly interacts with the Onuc atom. This large proximity of both atoms is probably the reason of the high positive charge accumulation in the Onuc from the reactant to TS states (0.07 a.u., which is 0.05 a.u. higher than the one obtained here for the NtMGAM). The NtMGAM, α-amylase and β-galactosidase enzymes have a conserved Arg (Arg526, Arg195 and Arg388, respectively) near the nucleophilic residue. The proximity order between the arginine and the carboxylic nucleophile group is NtMGAM (4.45 Å) < α-amylase (4.54 Å) < β-galactosidase (4.59 Å). The CBHI does not have any positive charged residue near to the Onuc atom, which could justify the lesser dissociative character of the TS. The current TS1 also differs to those obtained for α-mannosidase II17, β-galactosidase26, cellulase Cel7A20, β-hexosaminidase A21, cellulase Cel5A23, O-GlcNAcase25 and 1-3,1-4-β-glucanase28, in which the transition state geometry has the glycosidic bond almost broken, but the proton from the acid/base residue has not been transferred yet. Previous studies suggested that the other oxygen atom of the nucleophile residue establishes an important H-bond with the hydroxyl group bound to C2 atom of gluc-1 (C2-OH). Specifically, and for β-glycosidases’ retaining enzymes, it was verified that it stabilizes the sugar ring conformation and decreases the barrier required to attain the products37,

53

. However, in the present reaction

mechanism of NtMGAM, we verified that this short bond only happens in the reactants and TS geometries (1.73 Å and 1.81 Å lengths, respectively). This value increases to 3.99 Å in the covalent glycosyl-enzyme intermediate geometry due to the rotation of the C2-OH group of gluc-1 toward to Arg526 and Asp542 (now deprotonated) with lengths of 2.67 Å and 1.83 Å, respectively. A similar behavior was observed in the glycosylation reaction of CBHI, in which the C2-OH – nucleophile interaction was lost in some TS and product geometries with E3 and 4C1 sugar ring conformations,


15


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respectively27.

Deglycosylation step (hydrolysis) At the end of the first mechanistic step, and to complete the hydrolysis reaction, the gluc-2 molecule diffuses and dissociates from the active site, whilst one incoming water molecule approximates from the bulk solvent. To simulate these changes, we have considered a different QM region for the second step through the removal of the gluc-2 unit and the inclusion of one water molecule and the Asp203 residue that establishes a short H-bond with this solvent unit (see the geometry of this second intermediate (INT2) in Fig. 3a). In the second mechanistic step (deglycosylation step), the deprotonated side chain of Asp542 acted as a base catalyst accepting one proton from the incoming water molecule. This, in turn, acted as a nucleophile that attacks the C1 atom and displaces the carboxylate side chain of Asp443, yielding the final products with overall retention of stereochemistry. The glycosyl-NtMGAM intermediate, transition state (TS2) and product geometries of this step are illustrated in Figures 3a, 3b and 3c, respectively.


16

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Figure 3 – Illustration of the second intermediate (INT2), transition state (TS2) and product (P) geometries of the deglycosylation reaction. The main interactions established are highlighted. Only the polar hydrogen atoms are depicted.

In the TS2, the Asp443 side chain is completely displaced from the sugar (2.96 Å), whilst the glycosidic bond is not fully formed (Owat-C1 of 1.68 Å). From these geometrical parameters, it is clear that like TS1, the TS2 is also very loose structurally, and it has the expected dissociative nature. Additionally, the proton from the water molecule comes close to the general base Asp542, but it has not been transferred yet (1.40 Å). The fact that the C2-OH group establishes a short Hbond with the Asp542 catalyst (1.85 Å) probably makes this H transfer to take place at a later stage. A distortion on sugar ring conformation was also observed throughout this step, as seen in Figure S4b in SI. Although the results of this second mechanistic step look comparable to those obtained for other glycosidases, they differ on the distance between the anomeric carbon C1 and the nucleophilic water. The TS2 of NtMGAM has a much smaller C1-Owat distance than the C1-O-Asp443 distance (difference of 1.28 Å). A high difference between the two distances was also observed in the reaction of the cellulase Cel7A20 (difference of 1.12 Å). However, the TS2 of NtMGAM has a much stronger

dissociative

character

than

those

of

α-amylase22,


cellulase

Cel5A23,

N-

17


Page 18 of 28

acetylglucosaminidase30 and β-galactosidase26. The same was observed for the first step as well.

Energetic pathway of the NtMGAM catalytic mechanism The activation and reaction free energies of both mechanistic steps were calculated at the ONIOM(B1B95/6-311+G(2d,2p):Amber//B3LYP/6-31G(d):Amber) level of theory. Figure 4 displays the obtained energetic profile.

Figure

4

–

Energetic

path

at

the

ONIOM(B1B95/6-311+G(2d,2p):Amber//B3LYP/6-

31G(d):Amber) level of theory for the entire catalytic mechanism of the human NtMGAM. According to these results, the barriers of the first and second mechanistic steps are 17.6 kcal.mol-1 and 15.8 kcal.mol-1, respectively. Both values agree with the experimental activation energy, as the kcat for human NtMGAM is of 48.6 s-1, which corresponds to a barrier of 15.8 kcal.mol-1.8 They differ in up to 1.8 kcal.mol-1, which is within the range of the error used by the current DFT approach. The obtained reaction free energies of both steps were -8.0 kcal.mol-1 and -1.6 kcal.mol-1.


18

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In addition, the active site conformation could rearrange between the two mechanistic steps due to the dissociation of the glucose molecule and/or the displacement of some solvent molecules. However, it is not completely clear that the reaction cannot proceed with the glucose inside the active pocket. As the correct order of these events is unknown, we have considered a discontinuity between the two steps in the free energy profile for the reaction. Considering that TS1 has the highest energy, the glycosylation step should correspond to the rate-limiting step. However, given the proximity of the two barriers (1.8 kcal.mol-1), we consider that both contributions are equally relevant for the rate and kinetics of the NtMGAM catalytic mechanism. This is expectable due to the similar reaction catalyzed in the two steps. Both mechanistic steps are exoergonic. Single-point calculations were also performed using different density functionals. Table 1 displays the activation and reaction free energies obtained with these calculations. Table 2. Activation and reaction free energies attained with six different density functionals, throughout the NtMGAM catalytic mechanism. Thermal correction to enthalpy and entropy values were also shown. All values are in kcal.mol-1. density functional B1B95

TS1 17.6

INT -8.0

TS2 15.8

P -1.6

MPW1B95 CAM-B3LYP

18.8 18.9

-7.4 -5.7

16.1 15.0

-1.8 -1.2

PBE1PBE wB97X-D

18.1 20.7

-6.8 -4.3

15.2 16.6

-0.4 -1.3

BB1K corrections

20.5

-7.5

17.0

-2.3

Thermal correction to Enthalpy Entropy

-1.8 0.7

-1.1 -0.6

-2.6 0.6

-0.6 -2.2

As seen in Table 2, the activation free energies obtained for the first mechanistic step range from 17.6 kcal.mol-1 (B1B95 density functional) to 20.7 kcal.mol-1 (wB97X-D density functional), whilst they vary from 15.0 kcal.mol-1 (CAM-B3LYP density functional) to 17.0 kcal.mol-1 (BB1K density


19


Page 20 of 28

functional) in the second step. The maximum variations of the reaction free energies are 3.7 kcal.mol-1 and 1.9 kcal.mol-1 for the first and second mechanistic steps, respectively. Hence, the impact and energy dependence that different density functionals have on the glycosylation step is higher than on the deglycosylation step.

The vibrational entropy corrections shown in Table 2 were calculated using the harmonic oscillator approximation. However, we also determined these values using the combined free-rotor/harmonic oscillator approximation for a threshold vibrational temperature of 120 K (~100 cm-1) as proposed by Grimme et al.50 (see the results in Table S3). The difference between the two approximations is quite small (mostly < 1kcal/mol). As already mentioned, the establishment of a short H-bond between the C2-OH and one oxygen atom of the nucleophile residue has been associated with a decrease in the activation free energies throughout the retaining glycosidase reaction mechanisms37, 53. In our results, we only observed this H-bond interaction in the reactants and TS1 geometries. In order to evaluate the influence of this interaction on the barrier of the second step, we have modelled the covalent glycosyl-enzyme intermediate through manual rotation of the C2-OH, forcing it to face the oxygen atom of the nucleophile Asp443. The PES of the deglycosylation step was re-calculated using this geometry as starting structure. Figure S5 in SI displays the reactant (INT2’), transition state (TS2’) and product (P’) geometries for this alternative pathway. The energetic pathway of this substitute mechanistic step at the ONIOM(B1B95/6-311+G(2d,2p):Amber//B3LYP/6-31G(d):Amber) level of theory was determined. The obtained activation and reaction free energies were 10.1 kcal.mol-1 and -13.2 kcal.mol-1, respectively. However, comparing the energies of the two covalent glycosyl-enzyme intermediates, the one without the C2-OH-Asp443 H-bond is energetically more favored by 10.5 kcal.mol-1. This means that the barrier is 20.6 kcal.mol-1, which is 4.8 kcal mol-1 higher than the reaction with no H-bond. This data indicates that the presence of this hydrogen bridge is not


20

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essential, nor it favors the α-glycosidase NtMGAM catalytic mechanism. Furthermore, the values of the key distances Hwat-Asp542, Owat-C1, Onuc-C1 are quite similar for both alternatives of the deglycosylation step. This means that the C2-OH-Onuc hydrogen bond does not catalyse the chemical reaction of this step.

Conclusions To provide new valuable insights into glycosidic linkages cleavage reactions, we uncovered the atomic details of the catalytic mechanism of the human intestinal α-glycosidase (NtMGAM subunit) using a multi-resolution QM/MM computational approach. We have employed the ONIOM

method,

described

at

the

ONIOM(B1B95/6-311+G(2d,2p):Amber//B3LYP/6-

31G(d):Amber) level of theory. Considering that it is a crucial enzyme for increasing the postprandial glucose blood levels, the present results could have significant impact for inhibiting its activity by structure-based drug design strategies. Our calculations provide strong support for Koshland’s retaining mechanism, in which both mechanistic steps follow an SN2-like mechanism via loose transition states. The obtained TS1 geometry shows the glycosidic bond completely broken, the glycosidic oxygen already protonated by the Asp542, with the C1-Onuc bond not yet formed. The glycosylation step generated a stable covalent glycosyl-NtMGAM intermediate, which was later hydrolyzed in the second (deglycosylation) step. The TS2 geometry has the nucleophile Asp443 already displaced from the sugar, the proton from the incoming water molecule has not been transferred yet to the general base Asp542, and the new glycosidic bond is not fully formed. In both steps, a distortion of the pyranose ring of gluc-1 at the TSs is observed, in line with the planarity of the C5-O5-C1-C2 dihedral angle and the shorter C1-O5 bond and. All these geometrical parameters support the oxocarbenium-like


21


Page 22 of 28

character of the TSs. Additionally, both reaction steps exhibit TS with high dissociative character, being clearly more dissociative than those of α-amylase, a GH that catalyzes an identical reaction.

In addition to the catalytic acid/base and nucleophilic residues, several other residues were identified or confirmed as relevant for the maintenance of sugar conformations throughout the entire catalytic pathway. The Asp327, Arg526 and His600 residues establish H-bonds with the sugar hydroxyl groups, while the side chain rings of the Tyr214, Tyr299, Trp406, Phe450, Trp559 and Phe575 residues have stacking contacts with the sugar rings. The two Asp366 and Tyr214 residues also have a key role to modulate the pKa values of the acid/base and nucleophilic catalytic residues. We have found that the activation free energies of both mechanistic steps at the ONIOM(B1B95/6311+G(2d,2p):Amber//B3LYP/6-31G(d):Amber) level of theory are 17.6 kcal.mol-1 and 15.8 kcal.mol-1, respectively. We concluded that despite the glycosylation step seems to be the ratelimiting step, the small difference between the two barriers (1.8 kcal.mol-1) suggest that both could contribute for the rate of the entire reaction. Additionally, both barriers are in good agreement with the experimental data. The obtained reaction free energies of both steps were -8.0 kcal.mol-1 and 1.6 kcal.mol-1, respectively. Both mechanistic steps are exothermic, when considered individually. Single-point calculations with other density functionals revealed that the glycosylation step is energetically more dependent to the choice of the density functional, and that the barrier values of the second step are closer to the experimental data and within the error of the method. We also verified that the H-bond between the C2-OH of gluc-1 and the oxygen atom of the nucleophilic Asp542 residue increases the barrier of the second mechanistic step by 4.8 kcal mol-1. Hence, we conclude that the presence of this H-bridge is unfavorable and not essential for the NtMGAM catalytic mechanism. Our overall results improve the mechanistic and structural knowledge of the catalytic pathways of


22

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glycosidic bonds cleavage, which is very useful for drug design studies.

Supporting Information Superimposition of the active site residues of the NtMGAM and NtMGAM:acarbose X-ray structures (Figure S1), superimposition of the reactant geometry optimized with B3LYP and B3LYP-D3 (Figure S2), RMSd values during the MD simulation (Figure S3), superimposition of the glucose ring conformations (Figure S4), reactant, TS and product states of an alternative deglycosylation step (Figure S5), list of the QM atoms (Table S1), H-bonds between the NtMGAM and the substrate during the MD simulation (Table S2), activation and reaction free energies obtained with the combined free-rotor/harmonic oscillator approximation (Table S3), geometries of all the stationary points.

Acknowledgements This work received financial support from FEDER funds (POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 - UID/MULTI/04378/2013; NORTE-01-0145FEDER-000024, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, within the European Regional Development Fund. Natércia F. Brás acknowledges the support of the FCT through her IF starting grant (IF/01355/2014).

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TOC Graphic


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Mechanistic Pathway on Human Î±-Glucosidase Maltase

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