QM/MM study of the catalytic reaction of aphid myrosinase.

Brevicoryne brassicae, an aphid species, exclusively consumes plants from the Brassicaceae family and employs a sophisticated defense mechanism involving a myrosinase enzyme that breaks down glucosinolates obtained from its host plants. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations to study the catalytic reaction of aphid myrosinase. A proper QM region to study the myrosinase reaction should contain the whole substrate, models of Gln-19, His-122, Asp-124, Asn-166, Glu-167, Lys-173, Tyr-180, Val-228, Tyr-309, Tyr-346, Ile-347, Glu-374, Glu-423, Trp-424, and a water molecule. The calculations show that Asp-124 and Glu-423 must be charged, His-122 must be protonated on NE2, and Glu-167 must be protonated on OE2. Our model reproduces the anomeric retaining characteristic of myrosinase and indicates that the deglycosylation reaction is the rate-determining step of the reaction. Based on the calculations, we propose a reaction mechanism for aphid myrosinase-mediated hydrolysis of glucosinolates with an overall barrier of 15.2 kcal/mol. According to the results, removing a proton from Arg-312 or altering it to valine or methionine increases glycosylation barriers but decreases the deglycosylation barrier.

Two local minima for structures of [4Fe-4S] clusters obtained with density functional theory methods.

[4Fe-4S] clusters are essential cofactors in many proteins involved in biological redox-active processes. Density functional theory (DFT) methods are widely used to study these clusters. Previous investigations have indicated that there exist two local minima for these clusters in proteins. We perform a detailed study of these minima in five proteins and two oxidation states, using combined quantum mechanical and molecular mechanical (QM/MM) methods. We show that one local minimum (L state) has longer Fe-Fe distances than the other (S state), and that the L state is more stable for all cases studied. We also show that some DFT methods may only obtain the L state, while others may obtain both states. Our work provides new insights into the structural diversity and stability of [4Fe-4S] clusters in proteins, and highlights the importance of reliable DFT methods and geometry optimization. We recommend r2SCAN for optimizing [4Fe-4S] clusters in proteins, which gives the most accurate structures for the five proteins studied.

Catalytic Reaction Mechanism of Glyoxalase II: A Quantum Mechanics/Molecular Mechanics Study.

Methylglyoxal (MG) is a reactive and toxic compound produced in carbohydrate, lipid, and amino acid metabolism. The glyoxalase system is the main detoxifying route for MG and consists of two enzymes, glyoxalase I (GlxI) and glyoxalase II (GlxII). GlxI catalyzes the formation of S-d-lactoylglutathione from hemithioacetal, and GlxII converts this intermediate to d-lactate. A relationship between the glyoxalase system and some diseases like diabetes has been shown, and inhibiting enzymes of this system may be an effective means of controlling certain diseases. A detailed understanding of the reaction mechanism of an enzyme is essential to the rational design of competitive inhibitors. In this work, we use quantum mechanics/molecular mechanics (QM/MM) calculations and energy refinement utilizing the big-QM and QM/MM thermodynamic cycle perturbation methods to propose a mechanism for the GlxII reaction that starts with a nucleophilic attack of the bridging OH- group on the substrate. The coordination of the substrate to the Zn ions places its electrophilic center close to the hydroxide group, enabling the reaction to proceed. Our estimated reaction energies are in excellent agreement with experimental data, thus demonstrating the reliability of our approach and the proposed mechanism. Additionally, we examined alternative protonation states of Asp-29, Asp-58, Asp-134, and the bridging hydroxide ion in the catalytic process. However, these give less favorable reactions, a poorer reproduction of the crystal structure geometry of the active site, and higher root-mean-squared deviations of the active site residues in molecular dynamics simulations.

Benchmark Study of Redox Potential Calculations for Iron-Sulfur Clusters in Proteins.

Redox potentials have been calculated for 12 different iron-sulfur sites of 6 different types with 1-4 iron ions. Structures were optimized with combined quantum mechanical and molecular mechanical (QM/MM) methods, and the redox potentials were calculated using the QM/MM energies, single-point QM methods in a continuum solvent or by QM/MM thermodynamic cycle perturbations. We show that the best results are obtained with a large QM system (∼300 atoms, but a smaller QM system, ∼150 atoms, can be used for the QM/MM geometry optimization) and a large value of the dielectric constant (80). For absolute redox potentials, the B3LYP density functional method gives better results than TPSS, and the results are improved with a larger basis set. However, for relative redox potentials, the opposite is true. The results are insensitive to the force field (charges of the surroundings) used for the QM/MM calculations or whether the protein and solvent outside the QM system are relaxed or kept fixed at the crystal structure. With the best approach for relative potentials, mean absolute and maximum deviations of 0.17 and 0.44 V, respectively, are obtained after removing a systematic error of -0.55 V. Such an approach can be used to identify the correct oxidation states involved in a certain redox reaction.

Study of interaction of metal ions with methylthymol blue by chemometrics and quantum chemical calculations.

In this study, we determine the acidity constants of methylthymol blue (MTB) and association constants of its complexes with the ZnII, CuII, and FeII metal ions (MIs), through theoretical and experimental means. The complexes were characterized using UV-Visible absorption spectroscopy combined with soft/hard chemometrics methods and quantum chemical calculations. Quantum chemical calculations revealed that electronic transitions in the UV-Visible spectra of MTB have mixed n → π* and π → π* characters. The results of molar ratio and multivariate curve resolution alternating least squares (MCR-ALS) revealed the formation of successive 1:2 and 1:1 complexes (MI:MTB) for the ZnII and CuII systems. However, the formation of successive 1:1 and 2:1 complexes are suggested for FeII by the molar ratio and MCR-ALS. The majority of transitions observed in the UV-Visible spectra of the Zn(MTB) and Cu(MTB) complexes have ligand-to-ligand charge transfer (LLCT) characters. However, the transitions in the UV-Visible spectrum of the Fe(MTB) complex have LLCT and metal-to-ligand charge transfer (MLCT) characters. For the Fe2(MTB) complex, the lowest energy transition of has an LLCT character. However, its higher energy transitions are a mixture of LLCT, MLCT, and metal-to-metal charge transfer (MMCT) characters. The correlation between experimental and computed wavelengths revealed that the 1:1 complexes of ZnII and CuII prefer square pyramidal geometries. However, the FeII complexes always show octahedral geometry.

QM/MM Study of the Catalytic Reaction of Myrosinase; Importance of Assigning Proper Protonation States of Active-Site Residues.

Myrosinase from Sinapis alba hydrolyzes glycosidic bonds of ß-d-S-glucosides. The enzyme shows an enhanced activity in the presence of l-ascorbic acid. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations to study the catalytic reaction of wild-type myrosinase and its E464A, Q187A, and Q187E mutants. Test calculations show that a proper QM region to study the myrosinase reaction must contain the whole substrate, models of Gln-187, Glu-409, Gln-39, His-141, Asn-186, Tyr-330, Glu-464, Arg-259, and a water molecule. Furthermore, to make the deglycosylation step possible, Arg-259 must be charged, Glu-464 must be protonated on OE2, and His-141 must be protonated on the NE2 atom. The results indicate that assigning proper protonation states of the residues is more important than the size of the model QM system. Our model reproduces the anomeric retaining characteristic of myrosinase and also reproduces the experimental fact that ascorbate increases the rate of the reaction. A water molecule in the active site, positioned by Gln-187, helps the aglycon moiety of the substrate to stabilize the buildup of negative charge during the glycosylation reaction and this in turn makes the moiety a better leaving group. The water molecule also lowers the glycosylation barrier by ∼9 kcal/mol. The results indicate that the Q187E and E464A mutants but not the Q187A mutant can perform the glycosylation step. However, the energy profiles for the deglycosylation step of the mutants are not similar to that of the wild-type enzyme. The Glu-464 residue lowers the barriers of the glycosylation and deglycosylation steps. The ascorbate ion can act as a general base in the reaction of the wild-type enzyme only if the Glu-464 and His-141 residues are properly protonated.

Two-Substrate Glyoxalase I Mechanism: A Quantum Mechanics/Molecular Mechanics Study.

Glyoxalase I (GlxI) is an important enzyme that catalyzes the detoxification of methylglyoxal (MG) with the help of glutathione (H-SG). It is currently unclear whether MG and H-SG are substrates of GlxI or whether the enzyme processes hemithioacetal (HTA), which is nonenzymatically formed from MG and H-SG. Most previous studies have concentrated on the latter mechanism. Here, we study the two-substrate reaction mechanism of GlxI from humans (HuGlxI) and corn (ZmGlxI), which are Zn(II)-active and -inactive, respectively. Hybrid quantum mechanics/molecular mechanics calculations were used to obtain geometrical structures of the stationary points along reaction paths, and big quantum mechanical systems with more than 1000 atoms and free-energy perturbations were used to improve the quality of the calculated energies. We studied, on an equal footing, all reasonable reaction paths to the S- and R-enantiomers of HTA from MG and H-SG (the latter was considered in two different binding modes). The results indicate that the MG and H-SG reaction in both enzymes can follow the same path to reach S-HTA. However, the respective overall barriers and reaction energies are different for the two enzymes (6.1 and -9.8 kcal/mol for HuGlxI and 15.7 and -2.2 kcal/mol for ZmGlxI). The first reaction step to produce S-HTA is facilitated by a crystal water molecule that forms hydrogen bonds with a Glu and a Thr residue in the active site. The two enzymes also follow similar paths to R-HTA. However, the reactions reach a deprotonated and protonated R-HTA in the human and corn enzymes, respectively. The production of deprotonated R-HTA in HuGlxI is consistent with other theoretical and experimental works. However, our calculations show a different behavior for ZmGlxI (both S- and R-HTA can be formed in the enzyme with the alcoholic proton on HTA). This implies that Glu-144 of corn GlxI is not basic enough to keep the alcoholic proton. In HuGlxI, the two binding modes of H-SG that lead to S- and R-HTA are degenerate, but the barrier leading to R-HTA is lower than the barrier to S-HTA. On the other hand, ZmGlxI prefers the binding mode, which produces S-HTA; this observation is consistent with experiments. Based on the results, we present a modification for a previously proposed two-substrate reaction mechanism for ZmGlxI.

Quantum Mechanics/Molecular Mechanics Study of the Reaction Mechanism of Glyoxalase I.

Glyoxalase I (GlxI) is a member of the glyoxalase system, which is important in cell detoxification and converts hemithioacetals of methylglyoxal (a cytotoxic byproduct of sugar metabolism that may react with DNA or proteins and introduce nucleic acid strand breaks, elevated mutation frequencies, and structural or functional changes of the proteins) and glutathione into d-lactate. GlxI accepts both the S and R enantiomers of hemithioacetal, but converts them to only the S-d enantiomer of lactoylglutathione. Interestingly, the enzyme shows this unusual specificity with a rather symmetric active site (a Zn ion coordinated to two glutamate residues; Glu-99 and Glu-172), making the investigation of its reaction mechanism challenging. Herein, we have performed a series of combined quantum mechanics and molecular mechanics calculations to study the reaction mechanism of GlxI. The substrate can bind to the enzyme in two different modes, depending on the direction of its alcoholic proton (H2; toward Glu-99 or Glu-172). Our results show that the S substrate can react only if H2 is directed toward Glu-99 and the R substrate only if H2 is directed toward Glu-172. In both cases, the reactions lead to the experimentally observed S-d enantiomer of the product. In addition, the results do not show any low-energy paths to the wrong enantiomer of the product from neither the S nor the R substrate. Previous studies have presented several opposing mechanisms for the conversion of R and S enantiomers of the substrate to the correct enantiomer of the product. Our results confirm one of them for the S substrate, but propose a new one for the R substrate.

Higher Flexibility of Glu-172 Explains the Unusual Stereospecificity of Glyoxalase I.

Despite many studies during the latest two decades, the reason for the unusual stereospecificity of glyoxalase I (GlxI) is still unknown. This metalloenzyme converts both enantiomers of its natural substrate to only one enantiomer of its product. In addition, GlxI catalyzes reactions involving some substrate and product analogues with a stereospecificity similar to that of its natural substrate reaction. For example, the enzyme exchanges the pro- S, but not the pro- R, hydroxymethyl proton of glutathiohydroxyacetone (HOC-SG) with a deuterium from D2O. To find some clues to the unusual stereospecificity of GlxI, we have studied the stereospecific proton exchange of the hydroxymethyl proton of HOC-SG by this enzyme. We employed density functional theory and molecular dynamics (MD) simulations to study the proton exchange mechanism and origin of the stereospecificity. The results show that a rigid cluster model with the same flexibility for the two active-site glutamate residues cannot explain the unusual stereospecificity of GlxI. However, using a cluster model with full flexibility of Glu-172 or a larger model with the entire glutamates, extending the backbone into the neighboring residues, the results showed that there is no way for HOC-SG to exchange its protons if the alcoholic proton is directed toward Glu-99. However, if the hydroxymethyl proton instead is directed toward the more flexible Glu-172, we find a catalytic reaction mechanism for the exchange of the HS proton by a deuterium, in accordance with experimental findings. Thus, our results indicate that the special stereospecificity of GlxI is caused by the more flexible environment of Glu-172 in comparison to that of Glu-99. This higher flexibility of Glu-172 is also confirmed by MD simulations. We propose a reaction mechanism for the stereospecific proton exchange of the hydroxymethyl proton of HOC-SG by GlxI with an overall energy barrier of 15 kcal/mol.