Catalytic role of proton transfers in the formation of a tetrahedral adduct in a serine carboxyl peptidase (original) (raw)

A General Acid−Base Mechanism for the Stabilization of a Tetrahedral Adduct in a Serine−Carboxyl Peptidase: A Computational Study

Journal of the American Chemical Society, 2005

Sedolisins (serine-carboxyl peptidases) belong to a recently characterized family of proteolytic enzymes (MEROPS S53) that have a fold resembling that of subtilisin and a maximal activity at low pH. 1 This family includes the peptidase CLN2, 2 a human enzyme for which mutations in the encoding CLN2 gene lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. The defining features of the sedolisin family are a unique catalytic triad, 4,5 Ser-Glu-Asp (Ser278-Glu78-Asp82 for kumamolisin-As; see ), as well as the presence of an aspartic acid residue (Asp164 for kumamolisin-As) that replaces Asn155 of subtilisin, a residue that creates the oxyanion hole. The X-ray crystallographic and mutagenesis studies 4,5 demonstrated that the serine residue is the catalytic nucleophile, while the nearby Glu is likely to act as the general base to accept the proton from Ser and assist in the nucleophilic attack. A fundamental question for serine-carboxyl peptidases is whether these enzymes use the catalytic mechanism similar to that of classical serine proteases with simple replacements of certain catalytic residues so that they could be active at low pH. Here we demonstrate from quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations that this may not be the case. Unlike serine proteases that use the oxyanion-hole interactions to achieve the electrostatic stabilization of the tetrahedral intermediate and adduct, the members of the sedolisin family seem to stabilize the tetrahedral intermediate and adduct primarily through a general acid-base mechanism (i.e., similar to the mechanism proposed for aspartic proteases 6 ).

Published on Web 10/21/2005 A General Acid-Base Mechanism for the Stabilization of a Tetrahedral Adduct in a Serine-Carboxyl Peptidase: A Computational Study

2005

Sedolisins (serine-carboxyl peptidases) belong to a recently characterized family of proteolytic enzymes (MEROPS S53) that have a fold resembling that of subtilisin and a maximal activity at low pH. 1 This family includes the peptidase CLN2, 2 a human enzyme for which mutations in the encoding CLN2 gene lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. 3 The defining features of the sedolisin family are a unique catalytic triad, 4,5 Ser-Glu-Asp (Ser278-Glu78-Asp82 for kumamolisin-As; see Figure 1), as well as the presence of an aspartic acid residue (Asp164 for kumamolisin-As) that replaces Asn155 of subtilisin, a residue that creates the oxyanion hole. The X-ray crystallographic and mutagenesis studies4,5 demonstrated that the serine residue is the catalytic nucleophile, while the nearby Glu is likely to act as the general base to accept the proton from Ser and assist in the nucleophilic attack. A fundamental question for

Clarification of the mechanism of acylation reaction and origin of substrate specificity of the serine-carboxyl peptidase sedolisin through QM/MM free energy simulations

2011

Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzymecatalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P 1 site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers as well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P 1 site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P 1 site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N-H of P 1 0-Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P 1-Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P 1-Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P 1-Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton transfers during acylation are also given.

Energy Compensation Mechanism for Charge-Separated Protonation States in Aspartate−Histidine Amino Acid Residue Pairs

The Journal of Physical Chemistry B, 2010

The initial stage of proton propagation in the D-path channel of bovine cytochrome c oxidase, consisting of the approach of an H + to the entrance of this specific pathway, is inspected via first-principles calculations. Our model, extracted from the X-ray crystallographic structure, includes the amino acid residue pair aspartate (Asp91) and histidine (His503) as protonatable sites. Our calculations show that an additional proton, corresponding to the H + uptake by the enzyme from the inner bulk water, is transferred to either Asp91 or His503, leading to the formation of a neutral or a charge-separated protonation state. The relative stability between the two states amounts to a total energy difference of about 5 kcal/mol; this indicates that both Asp91 and His503 are involved in the proton supply to the D-path, playing the role of proton acceptors. The hydrogen-bond environment around Asp91 and His503 has an important influence on both the energetics and the electronic structure of the system; for instance, it compensates the Coulomb-energy cost in the chargeseparated protonation state. An energy partitioning analysis shows that the compensatory effect is mainly due to local electrostatic interactions among the charged Asp91 and His503 side chains and the surrounding polar residues. The energy compensation mechanism we found in this work balances the energetics of Asp-His pairs, hence permitting an efficient and selective regulation of the protonatable amino acid residues, where several protonation states are accessible within energy differences of the order of a few H-bonds.

The QM/MM Molecular Dynamics and Free Energy Simulations of the Acylation Reaction Catalyzed by the Serine-Carboxyl Peptidase Kumamolisin-As †

Biochemistry, 2007

Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations are performed to study the acylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism and the origin of substrate specificity. It is demonstrated that the nucleophilic attack by the serine residue on the substrate may not be the rate-limiting step for the acylation of the GPH*FF substrate. The present study also confirms the earlier suggestions that Asp164 acts as a general acid during the catalysis and that the electrostatic oxyanion hole interactions may not be sufficient to lead a stable tetrahedral intermediate along the reaction pathway. Moreover, Asp164 is found to act as a general base during the formation of the acyl-enzyme from the tetrahedral intermediate. The role of dynamic substrate assisted catalysis (DSAC) involving His at the P 1 site of the substrate is examined for the acylation reaction. It is demonstrated that the bond-breaking and -making events at each stage of the reaction trigger a change of the position for the His side chain and lead to the formation of the alternative hydrogen bonds. The back and forth movements of the His side chain between the CdO group of Pro at P 2 and O δ2 of Asp164 in a ping-pong-like mechanism and the formation of the alternative hydrogen bonds effectively lower the free energy barriers for both the nucleophilic attack and the acyl-enzyme formation and may therefore contribute to the relatively high activity of kumamolisin-As toward the substrates with His at the P 1 site.

Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5′-phosphate dependent enzymes l-dopa decarboxylase and alanine racemase

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011

Molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential have been carried out to investigate the internal proton transfer equilibrium of the external aldimine species in L-dopa decarboxylase, and carbanion stabilization by the enzyme cofactor in the active site of alanine racemase. Solvent effects lower the free energy of the O-protonated PLP tautomer both in aqueous solution and in the active site, resulting a free energy difference of about-1 kcal/mol relative to the N-protonated Schiff base in the enzyme. The external aldimine provides the dominant contribution to lowering the free energy barrier for the spontaneous decarboxylation of L-dopa in water, by a remarkable 16 kcal/mol, while the enzyme L-dopa decarboxylase further lowers the barrier by 8 kcal/mol. Kinetic isotope effects were also determined using a path integral free energy perturbation theory on the primary 13 C and the secondary 2 H substitutions. In the case of alanine racemase, if the pyridine ring is unprotonated as that in the active site, there is destabilizing contribution to the formation of thecarbanion in the gas phase, although when the pyridine ring is protonated the contribution is stabilizing. In aqueous solution and in alanine racemase, the-carbanion is stabilized both when the pyridine ring is protonated and unprotonated. The computational studies illustrated in this article show that combined QM/MM simulations can help provide a deeper understanding of the mechanisms of PLP-dependent enzymes. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases

Proceedings of the National Academy of Sciences, 1975

The charge relay system and its role in the acylation of serine proteinases is studied using the partial retention of diatomic differential overlap (PRDDO) technique to perform approximate ab initio molecular orbital calculations on a model of the enzyme-substrate complex. The aspartate in the charge relay system is seen to act as the ultimate proton acceptor during the charging of the serine nucleophile. A projection of the potential energy surface is obtained in a subspace corresponding to this charge transfer and to the coupled motions of active site residues and the substrate. These results together with extended basis set results for cruder models suggest that a concerted transfer of protons from Ser-195 to His-57 and from His-57 to Asp-102 occurs with an energy barrier of 20-25 kcal/mole (84-105 kJ/ mole). The subsequent nucleophilic attack on the scissile peptide linkage by the charged serine is then seen to proceed energetically downhill to the tetrahedral intermediate. The formation of the tetrahedral intermediate from the Michaelis complex is calculated to be nearly thermoneutral. The serine proteinases [e.g., chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4)] catalyze the hydrolysis of proteins during digestion in many animals and have been the subject of much research. A considerable body of evidence has accumulated which suggests that chymotrypsin-mediated hydrolysis of many substrates proceeds via the mechanism kk.

Assigning the Protonation States of the Key Aspartates in β-Secretase Using QM/MM X-ray Structure Refinement

Journal of Chemical Theory and Computation, 2006

β-Secretase, a.k.a. β-APP cleaving enzyme (BACE), is an aspartyl protease that has been implicated as a key target in the pathogenesis of Alzheimer's disease (AD). The identification of the protonation states of the key aspartates in β-secretase is of great interest both in understanding the reaction mechanism and in guiding the design of drugs against AD. However, the resolutions of currently available crystal structures for BACE are not sufficient to determine the hydrogen atom locations. We have assigned the protonation states of the key aspartates using a novel method, QM/MM X-ray refinement. In our approach, an energy function is introduced to the refinement where the atoms in the active site are modeled by quantum mechanics (QM) and the other atoms are represented by molecular mechanics (MM). The gradients derived from the QM/MM energy function are combined with those from the X-ray target to refine the crystal structure of a complex containing BACE and an inhibitor. A total number of 8 protonation configurations of the aspartyl dyad were considered and QM/MM X-ray refinements were performed for all of them. The relative stability of the refined structures was scored by constructing the thermodynamic cycle using the energetics calculated by fully quantum mechanical self-consistent reaction field (QM/SCRF) calculations. While all 8 refined structures fit the observed electron density about equally well, we find the mono-protonated configurations to be strongly favored energetically, especially the configuration with the inner oxygen of Asp32 protonated and the hydroxyl of the inhibitor pointing towards Asp228. It was also found that these results depend on the constraints imposed by the X-ray data. We suggest that one of the strengths of this approach is that the resulting structures are a consensus of theoretical and experimental data and remark on the significance of our results in structure based drug design and mechanistic studies.

Energy barriers of proton transfer reactions between amino acid side chain analogs and water fromab initio calculations

Journal of Computational Chemistry, 2006

Proton transfer reactions were studied in all titratable pairs of amino acid side chains where, under physiologically reasonable conditions, one amino acid may function as a donor and the other one as an acceptor. Energy barriers for shifting the proton from donor to acceptor atom were calculated by electronic structure methods at the MP2/6-31þþG(d,p) level, and the well-known double-well potentials were characterized. The energy difference between both minima can be expressed by a parabola using as argument the donor-acceptor distance R(DA). In this work, the fit parameters of the quadratic expression are determined for each donor-acceptor pair. Moreover, it was found previously that the energy barriers of the reactions can be expressed by an analytical expression depending on the distance between donor and acceptor and the energy difference between donor and acceptor bound states. The validity of this approach is supported by the extensive new data set. This new parameterization of proton transfer barriers between titratable amino acid side chains allows us to very efficiently estimate proton transfer probabilities in molecular modelling studies or during classical molecular dynamics simulation of biomolecular systems.

The efficiency of proton transfer in Kirby’s enzyme model, a computational approach

Tetrahedron Letters, 2010

DFT and ab initio calculation results for proton transfer reactions in Kirby's acetals reveal that the mechanism proceeds via efficient intramolecular general acid catalysis (IGAC) and not through a 'classical' general acid catalysis mechanism (GAC). Further, they show that the driving force for the proton transfer efficiency is the proximity of the two reactive centers (r) and the attack angle (a), and the rate of the reaction is linearly correlated with r 2 and sin (180°À a). Acetals with short r values and with a values close to 180°(forming a linear H-bond) are more reactive due to the development of strong hydrogen bonds in their global minimum, transition state, and product structures.