Kana Kobayashi

Modeling the Enolization of Succinimide Derivatives, a Key Step of Racemization of Aspartic Acid Residues: Importance of a Two‐H2O Mechanism

Racemization of aspartic acid residues in peptides and proteins is assumed to proceed via succinimide intermediates. An enolization of the succinimide intermediate is required for the racemization to occur. In this study, we modeled the enolization step by density‐functional theory (DFT) calculations (B3LYP/6‐31+G**), using two model compounds, N‐methylsuccinimide (1) and its formylamino derivarive 2. Three mechanisms were investigated for 1, i.e., the direct mechanism without active participation of H2O molecules, and one‐H2O and two‐H2O mechanisms, in which one or two H2O molecules actively participate in the reaction. We found that the two‐H2O mechanism was the most favorable with an activation barrier of 37 kcal mol−1. In the two‐H2O mechanism, a concerted bond reorganization involving a triple H‐atom transfer occurred in an eight‐membered cyclic structure formed between the imide and two H2O molecules. For 2, we investigated only the two‐H2O mechanism and found that the activation barrier was lowered to 31 kcal mol−1 due to an H‐bond between the CO O‐atom of the formylamino group (‘the neighboring residue’) and one of the H2O molecules. Our results suggest that, in proteins, the Asp racemization is severely controlled by the accessibility of H2O molecules to the reaction site of the succinimide intermediate.

Evaluation of Influence of Single Nucleotide Polymorphisms in Cytochrome P450 2B6 on Substrate Recognition Using Computational Docking and Molecular Dynamics Simulation

In this study, we investigated the influence of single nucleotide polymorphisms on the conformation of mutated cytochrome P450 (CYP) 2B6 proteins using molecular dynamics (MD) simulation. Some of these mutations influence drug metabolism activities, leading to individual variations in drug efficacy and pharmacokinetics. Using computational docking, we predicted the structure of the complex between the antimalarial agent artemether and CYP2B6 whose conformations were obtained by MD simulation. The simulation demonstrated that the entire structure of the protein changes even when a single residue is mutated. Moreover, the structural flexibility is affected by the mutations and it may influence the enzyme activity. The results suggest that some of the inactive mutants cannot recognize artemether due to structural changes caused by the mutation.

Comparison of molecular dynamics simulation methods for amyloid β1–42 monomers containing d-aspartic acid residues for predicting retention times in chromatography

Molecular dynamics simulations of amyloid β(1-42) containing D-aspartic acid residues were performed using several continuous solvent models to investigate the usefulness of simulation methods for D-amino acid-containing proteins and peptides. Normal molecular dynamics simulations and replica exchange molecular dynamics simulations, which are one of the generalized-ensemble algorithms, were performed. Because the β-structure contents of amyloid β(1-42) peptides obtained by replica exchange molecular dynamics simulations with Onufriev-Bashford-Case generalized Born implicit solvent were qualitatively consistent with experimental data, replica exchange molecular dynamics rather than other methods appeared to be more reasonable for calculations of amyloid β(1-42) containing D-aspartic acid residues. Computational results revealed that peptides with stereoinversion of Asp23 tend to form β-sheet structures by themselves, in contrast to the wild-type peptides that form β-sheet structures only after aggregation. These results are expected to be useful for computational investigations of proteins and peptides such as prediction of retention time of peptides and proteins containing D-aspartic acid residues.

Molecular‐Dynamics Simulations for Amyloid β1–42 Monomer with D‐Aspartic Acid Residues Using Continuous Solvent

Molecular‐dynamics simulations of amyloid‐β1–42 peptides including D‐aspartic acid residues were performed, and their three‐dimensional structures were compared. The simulations were performed in an aqueous environment using a continuous solvent model. In the structures obtained from simulations, the occurrence ratio of β‐extended structures for the peptide that included D‐Asp23 was larger than that for the wild‐type peptide. These β‐extended structures appeared in the C‐terminal region of the peptide, and the α‐helix structures of the region were lost. On the other hand, for the peptide that included the stereo‐inverted form of Asp1 as well as D‐Asp23, the occurrence ratio of β‐extended structures in the C‐terminal region was lower than that of the peptide including only D‐Asp23.

Computational insight into the mechanism of serine residue racemization.

Despite the known racemization of serine (Ser) residues in proteins from aged or diseased human brains, the mechanism of Ser racemization in peptides and proteins has not been studied. This is in contrast to the case of the rapid racemization of aspartic acid (Asp) residues, for which a succinimide‐mediated mechanism is established. In a possible mechanism for Ser racemization, the enolization involving the H(α)‐atom and the CO(α) group occurs by active participation of two H2O molecules. In this study, we computationally modeled this two‐H2O mechanism of enolization for Ser and alanine (Ala) residues using model compounds, and the results were compared with each other and with the case of succinimide analogues reported in a separated paper. Our results suggest that Ser residues are much more racemization‐prone than Ala residues, but to a lesser extent than Asp residues. The side‐chain CO bond of the Ser residue stabilizes the enolization transition state hyperconjugatively.

Computational investigation of the substrate recognition mechanism of protein d-aspartyl (l-isoaspartyl) O-methyltransferase by docking and molecular dynamics simulation studies and application to interpret size exclusion chromatography data☆

Unusual amino acid residues such as L-β-aspartyl (Asp), D-α-Asp, and D-β-Asp have been detected in proteins and peptides such as α-crystallin in the lens and β-amyloid in the brain. These residues increase with age, and hence they are associated with age-related diseases. The enzyme protein D-aspartyl (L-isoaspartyl) O-methyltransferase (PIMT) can revert these residues back to the normal L-α-Asp residue. PIMT catalyzes transmethylation of S-adenosylmethionine to L-β-Asp and D-α-Asp residues in proteins and peptides. In this work, the substrate recognition mechanism of PIMT was investigated using docking and molecular dynamics simulation studies. It was shown that the hydrogen bonds of Ser60 and Val214 to the carboxyl group of Asp are important components during substrate recognition by PIMT. In addition, specific hydrogen bonds were observed between the main chains of the substrates and those of Ala61 and Ile212 of PIMT when PIMT recognized L-β-Asp. Hydrophobic interactions between the (n-1) residue of the substrates and Ile212 and Val214 of PIMT may also have an important effect on substrate binding. Volume changes upon substrate binding were also evaluated in the context of possible application to interpretation of size exclusion chromatography data.

Computational modeling of the enolization in a direct mechanism of racemization of the aspartic acid residue.

The rapid racemization of aspartic acid (Asp) residues in peptides and proteins is due mainly to the succinimide intermediate. However, there should be another mechanism for Asp racemization without intermediacy of the succinimide. The direct H‐atom abstraction from the C(α)‐atom that leads to the enol form of the Asp residue is one possibility. In another study, we have computationally predicted that the corresponding enolization in the succinimide intermediate occurs by assistance of two H2O molecules. In the present study, we, therefore, investigated the similar two‐H2O‐assisted enolization for an Asp‐containing model compound by the same computational method as before (B3LYP/6‐31+G**). Rather surprisingly, the activation barrier for the two‐H2O‐assisted enolization of the Asp residue (protonated form) was calculated to be almost equal to that for the corresponding succinimide. Therefore, an Asp residue is expected to be prone to enolization to almost the same degree as the corresponding succinimide form, and the ‘direct’ (i.e., non‐succinimide‐mediated) mechanism of Asp racemization may compete with the succinimide‐mediated mechanism.