Semiempirical simulation of a theta‐class glutathione S‐transferase‐catalyzed glutathione attack to the allelochemical DIMBOA

We evaluated by the semiempirical method PM3 possible mechanisms of a putative interaction between a cereal allelochemical, the cyclic hydroxamic acid 2,4‐dihydroxy‐7‐metoxy‐2H‐1,4‐benzoxazin‐3(4H)‐one (DIMBOA), and the tripeptide glutathione (GSH) inside the active site of a theta‐class glutathione S‐transferase. Based on a preliminary study of transition states from DIMBOA reactions with methanethiolate as a simple model of GSH, we investigated the roles of catalytic residues of the enzyme during nucleophilic additions of GSH to the carbonyl groups of DIMBOA and of its phenol/aldehyde isomer inside the active site model. Our results suggest that a tyrosine residue, Tyr113, makes the most important contributions for the catalytic mechanism. In the modeled reaction steps, Tyr113 behaves as a double hydrogen bond donor catalyst for nucleophilic additions of GSH to substrates: It initially helps stabilize the strongly nucleophilic reduced GSH with a hydrogen bond intermediated by a water molecule; during substrate approach, small conformational changes enable the residue to make a direct hydrogen bond to the substrate group that develop negative charge after addition of reduced GSH. © 2002 Wiley Periodicals, Inc.; Int J Quantum Chem, 2002     

Mechanism of the glutathione transferase-catalyzed conversion of antitumor 2-crotonyloxymethyl-2-cycloalkenones to GSH adducts

Human glutathione (GSH) transferase (hGSTP1-1) processes with similar kinetic efficiencies the antitumor agents 2-crotonyloxymethyl-2-cyclohexenone (COMC-6), 2-crotonyloxymethyl-2-cycloheptenone (COMC-7), and 2-crotonyloxymethyl-2-cyclopentenone (COMC-5) to 2-glutathionylmethyl-2-cyclohexenone, 2-glutathionylmethyl-3-glutathionyl-2-cycloheptenone, and 2-glutathionylmethyl-2-cyclopentenone, respectively. This process likely involves initial enzyme-catalyzed Michael addition of GSH to the COMC derivative to give a glutathionylated enol(ate), which undergoes nonstereospecific ketonization, either while bound to the active site or free in solution, to a glutathionylated exocyclic enone. Free in solution, GSH reacts at the exomethylene carbon of the exocyclic enone, displacing the first GSH to give the final product. This mechanism is supported by the observation of multiphasic kinetics in the presence of high concentrations of hGSTP1-1 and the ability to trap kinetically competent exocyclic enones in aqueous acid using COMC-6 and COMC-7 as substrates. That the exocyclic enone is formed by nonstereospecific ketonization of an enol(ate) species is indicated by the observation that COMC-6 (chirally labeled with deuterium at the exomethylene carbon) gives stereorandomly labeled exocyclic enone. The isozymes hGSTP1-1, hGSTA1-1, hGSTA4-4, and hGSTM2-2 catalyze the conversion of COMC-6 to final product with similar efficiencies (K(m) = 0.08-0.34 mM, k(cat) = 1.5-6.1 s(-)(1)); no activity was detected with the rat rGSTT2-2 isozyme. Molecular docking studies indicate that in hGSTP1-1, the hydroxyl group of Tyr108 might serve as a general acid catalyst during substrate turnover. The possible significance of these observations with respect to the metabolism of COMC derivatives in multidrug resistant tumors is discussed.     

Organotellurium-bridged cyclodextrin dimers as artificial glutathione peroxidase models

To elucidate the importance of the goodness of fit in complexes between substrates and glutathione peroxidise (GPX) mimics, we examined the decomposition of a variety of structurally distinct hydroperoxides at the expense of glutathione (GSH) catalyzed by 2,2′-ditellurobis(2-deoxy-γ-cyclodextrin) (2-Te-γ-CD), and by the corresponding derivatives of β-cyclodextrin (β-CD) and α-cyclodextrin. The good fit of the cumene group into the γ-CD binding cavity reflected the result of well-defined reaction geometry, leading to the most excellent peroxidase activity with high substrate specificity. Furthermore, the catalytic constant and the combination with the best binding also exhibited the highest regioselectivity in the substrate decomposition. Saturation kinetics were observed and the catalytic reaction agreed with a ping-pong mechanism, in analogy with natural GPX, and might exert its thiol peroxidase activity via tellurol, tellurenic acid, and tellurosulfide. The stoichiometry of the inclusion complex was determined to be of 2:1 host-to-guest. The value of stability constant K _c for (2-Te-γ-CD)₂/GSH at room temperature was calculated to be 3.815?×?10⁴?M^?2, which suggested that 2-Te-γ-CD had a moderate ability to bind GSH. Importantly, the proposed mode of the (2-TeCD)₂/GSH complex was the possible important noncovalent interactions between enzymes and substrates in influencing catalysis and binding.     

Stereoselective alkylation–reduction of β-keto nitriles by the fungus Curvularia lunata

The alkylation–reduction (AR) reaction of β-keto nitriles by growing cells of Curvularia lunata CECT 2130 has been explored. The reaction conditions for the ethylation of benzoylacetonitrile have been optimized in terms of both yield and stereoselectivity and the mechanism of this biotransformation was studied. The scope of this reaction has been extended to other alkylations (R=Et, Pr, Bu, iso-Bu) and to a series of aromatic and heteroaromatic substrates, yielding the corresponding optically active α-alkyl β-hydroxy nitriles. The yield (directly related to competing carbonyl reduction reaction) depends on the substrate bulk, whereas the enantiomeric and diastereomeric excesses (both up to 98%) seem to be dependent on several factors.     

Structural and functional diversity of glutaredoxins in yeast

Glutaredoxins are defined as thiol disulfide oxidoreductases that reduce disulfide bonds employing reduced glutathione as electron donor. They constitute a complex family of proteins with a diversity of enzymatic and functional properties. Thus, dithiol glutaredoxins are able to reduce disulfide bonds and deglutathionylate mixed disulfides between glutathione and cysteine protein residues. They could act regulating the redox state of sulfhydryl residues of specific proteins, while thioredoxins (another family of thiol disulfide oxidoreductases which employ NADPH as electron donor) would be the general sulfhydryl reductants. Some dithiol glutaredoxins such as human Grx2 form dimers bridged by one iron-sulfur cluster, which acts as a sensor of oxidative stress, therefore regulating the activity of the glutaredoxin. The ability to interact with iron-sulfur clusters as ligands is also characteristic of monothiol glutaredoxins with a CGFS-type active site. These do not display thiol oxidoreductase activity, but have roles in iron homeostasis. The three members of this subfamily in Saccharomyces cerevisiae participate in the synthesis of the iron-sulfur clusters in mitochondria (Grx5), or in signalling the iron status inside the cell for regulation of iron uptake and intracellular iron relocalization (Grx3 and Grx4). Such a role in iron metabolism seems to be evolutionary conserved. Fungal cells also contain membrane-associated glutaredoxins structurally and enzymatically similar to dithiol glutaredoxins, which may act as redox regulators at the early stages of the protein secretory machinery.     

A Glutathione Peroxidase Mimic 6,6′-Ditellurobis (6-Deoxy-β-Cyclodextrin) with High Substrate Specificity

Glutathione peroxidase (GPx) is one of the most important antioxidative selenoenzymes in living organisms. The novel GPx mimic 6,6′-ditellurobis(6-deoxy-β-cyclodextrin) (6-TeCD) was prepared and evaluated for its capacity to catalyze the reduction of H₂O₂, tert-butyl hydroperoxide (t-BuOOH), and cumene hydroperoxide (CuOOH) by glutathione (GSH) or 3-carboxy-4-nitrobenzenethiol (ArSH). Compared the ArSH assay with the coupled reductase assay, we found that 6-TeCD exhibited strong substrate specificity for aromatic thiol substrate. The specificity led to efficient peroxidase activity almost 100,000-fold than that for a well-known GPx mimic diphenyl diselenide (PhSeSePh). Furthermore, reduction of lipophilic CuOOH was proceeded ca. 30 times faster than the more hydrophilic H₂O₂, which cannot bind into the hydrophobic cavity of β-cyclodextrin. Thus, it seemed that catalytic activity of cyclodextrin-derived GPx models strongly depends on the structurally different both substrates hydroperoxides (ROOH) and thiols.     

Mapping the hydrolytic and synthetic selectivity of a type C feruloyl esterase (StFaeC) from Sporotrichum thermophile using alkyl ferulates

The active site of Sporotrichum thermophile type C feruloyl esterase (StFaeC) was probed using a series of C₁–C₄ alkyl ferulates. The affinities for straight and branched alkyl ferulates were demonstrated by the K_m values of 1.64–0.51 and 0.19–0.1, respectively. Comparison of k_cat and k_cat/K_m values shows that the enzyme hydrolyzed n-propyl ferulate faster and iso-propyl ferulate more efficiently. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water–organic mixture consisting of n-hexane, t-butanol and water. Lengthening the aliphatic side chain was the most significant factor causing lower synthetic activity of the enzyme. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic process, such as the temperature and substrate concentration have been investigated. Under identical reaction conditions, the enzyme feruloylated other monosaccharides such as d-arabinose, d-glucose, d-xylose, d-mannose, d-fructose, d-galactose, d-ribose and model substrates such as 4-nitrophenyl α-l-arabinofuranoside and 4-nitrophenyl α-l-arabinopyranoside.     

The reaction mechanism of xanthine oxidase: evidence for two-electron chemistry rather than sequential one-electron steps

Current research on xanthine oxidase has favored a mechanism involving base-catalyzed proton abstraction from a Mo-OH group, allowing nucleophilic attack on the substrate and hydride transfer from the substrate to Mo=S group in the active site. During the course of this reaction mechanism, the molybdenum redox cycles from MoVI to MoIV, with reoxidation of the MoIV speices to form the EPR active MoV intermediate. However, it has also been suggested that the reaction occurs in two subsequent one-electron steps. We have determined kinetic parameters kred and kred/Kd for a variety of plausible substrates as well as the one-electron reduction potentials for these substrates. Our data indicate no correlation between these kinetic parameters and their one-electron reduction potentials, as would be expected if the enzyme were using two subsequent one-electron reduction steps. Our results provide additional support to current evidence for the favored two-electron reduction mechanism.     

Thermoprecipitation of Glutathione S‐Transferase by Glutathione–Poly(N‐isopropylacrylamide) Prepared by RAFT Polymerization

Herein, we report an effective and rapid method to purify glutathione S‐transferase (GST) using glutathione (GSH)‐modified poly(N‐isopropylacrylamide) (pNIPAAm) and mild, thermal conditions. A chain transfer agent modified with pyridyl disulfide was employed in the reversible addition–fragmentation chain transfer (RAFT) polymerization of NIPAAm. The resulting polymer had a narrow molecular weight distribution (polydispersity index = 1.21). Conjugation of GSH to the pyridyl disulfide–pNIPAAm reached 95% within 30 min as determined by UV–Vis monitoring of the release of pyridine‐2‐thione. GST was successfully thermoprecipitated upon heating the GSH–pNIPAAm above the lower critical solution temperature (LCST). The pull down assay was repeated with bovine serum albumin (BSA) and T4 lysozyme (T4L), which demonstrated the specificity of the polymer for GST. Due to its simplicity and high efficiency, this method holds great potential for large‐scale purification of GST‐tagged proteins.

     

Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency

Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides, and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes, and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. To fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved (11)CXX(14)C active site were chemically synthesized. Grx3, Grx3(C11U), and Grx3(C14U) exhibited redox potentials of -194, -260, and -275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (-309 mV) and thioredoxin (Trx) (-270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a "central atom" in the exchange reaction. The 10(2)-10(4)-fold increase in the rate of Trx reduction by the seleno-Grx3 analogues demonstrates that oxidoreductases containing either selenenyl-sulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts.     

A practical synthesis of optically active aromatic epoxides via asymmetric transfer hydrogenation of α-chlorinated ketones with chiral rhodium-diamine catalyst

A practical method for the synthesis of optically active aromatic epoxides has been developed via the formation of optically active α-chlorinated alcohols and intramolecular etherification. Optically active alcohols with up to 99% ee can be obtained from the asymmetric reduction of aromatic ketones with a substrate/catalyst ratio of 1000-5000 using a formic acid/triethylamine mixture containing a well-defined chiral Rh complex, Cp*RhCl[(R,R)-Tsdpen]. The asymmetric reduction of α-chlorinated aromatic ketones with a chiral Rh catalyst is characterized by a rapid and carbonyl group-selective transformation because of the coordinatively saturated nature of diamine-based Cp*Rh(III) hydride complexes. The outcome of the reduction is significantly influenced by the structures of the ketonic substrates as well as the hydrogen source such as formic acid or 2-propanol. Commercially available reagents and solvents can be used in this reaction without special purification. This epoxide synthetic process in either a one- or two-pot procedure is practical and particularly useful for the large-scale production of optically active styrene oxides from α-chlorinated ketones.     

Cyclodextrin-derived mimic of glutathione peroxidase exhibiting enzymatic specificity and high catalytic efficiency

To elucidate the relationships between molecular recognition and catalytic ability, we chose three assay systems using three different thiol substrates, glutathione (GSH), 3-carboxyl-4-nitrobenzenethiol (CNBSH), and 4-nitrobenzenethiol (NBSH), to investigate the glutathione peroxidase (GPx) activities of 2,2'-ditellurobis(2-deoxy-beta-cyclodextrin) (2-TeCD) in the presence of a variety of structurally distinct hydroperoxides (ROOH), H2O2, tert-butyl peroxide (tBuOOH), and cumene peroxide (CuOOH), as the oxidative reagent. A comparative study of the three assay systems revealed that the cyclodextrin moiety of the GPx mimic 2-TeCD endows the molecule with selectivity for ROOH and thiol substrates, and hydrophobic interactions are the most important driving forces in 2-TeCD complexation. Furthermore, in the novel NBSH assay system, 2-TeCD can catalyze the reduction of ROOH about 3.4 x 10(5) times more efficiently than diphenyl diselenide (PhSeSePh), and its second-order rate constants for thiol are similar to some of those of native GPx. This comparative study confirms that efficient binding of the substrate is essential for the catalytic ability of the GPx mimic, and that NBSH is the preferred thiol substrate of 2-TeCD among the chosen thiol substrates. Importantly, the proposed mode of action of 2-TeCD imitates the role played by several possible noncovalent interactions between enzymes and substrates in influencing catalysis and binding.     

Protein ubiquitination and formation of polyubiquitin chains without ATP,E1 and E2 enzymes

Studying protein ubiquitination is difficult due to the complexity of the E1–E2–E3 ubiquitination cascade. Here we report the discovery that C-terminal ubiquitin thioesters can undergo direct transthiolation with the catalytic cysteine of the model HECT E3 ubiquitin ligase Rsp5 to form a catalytically active Rsp5∼ubiquitin thioester (Rsp5∼Ub). The resulting Rsp5∼Ub undergoes efficient autoubiquitination, ubiquitinates protein substrates, and synthesizes polyubiquitin chains with native Ub isopeptide linkage specificity. Since the developed chemical system bypasses the need for ATP, E1 and E2 enzymes while maintaining the native HECT E3 mechanism, we named it “Bypassing System” (ByS). Importantly, ByS provides direct evidence that E2 enzymes are dispensable for K63 specific isopeptide bond formation between ubiquitin molecules by Rsp5 in vitro. Additionally, six other E3 enzymes including Nedd4-1, Nedd4-2, Itch, and Wwp1 HECT ligases, along with Parkin and HHARI RBR ligases processed Ub thioesters under ByS reaction conditions. These findings provide general mechanistic insights on protein ubiquitination, and offer new strategies for assay development to discover pharmacological modulators of E3 enzymes.     

Total synthesis and biochemical characterization of mirror image barnase

In this study we synthesized and characterized mirror image barnase (B. amyloliquefaciens ribonuclease). d-Barnase was identical to l-barnase, when analyzed by liquid chromatography and mass-spectrometry. Proteolysis of the mirror image enzyme revealed that in contrast to its native counterpart, d-barnase was completely stable to digestive proteases. In enzymatic assays, d-barnase had the reciprocal chiral specificity and was fully active towards mirror image substrates. Interestingly, d-barnase also hydrolyzed the substrate of the native chirality, albeit 4000 times less efficiently. This effect was further confirmed by digesting a native 112-mer RNA with the enzyme. Additional studies revealed that barnase accommodates a range of substrates with various chiralities, but the prime requirement for guanosine remains. These studies point toward using mirror image enzymes as modern agents in biotechnology.     

Comments on the paper of A.T. D'Agostino and P.N. Ross: “on the importance of surface structure in electrochemistry: the well-defined Au (111) single crystal electrode surface”

The electroreduction of dimethylpyrrolidinium (DMP⁺) in diglyme on mercury cathodes was investigated. It was found to proceed via a fast one-electron transfer resulting in an insoluble “amalgam”. The DMP⁺ amalgam was reactive and transferred electrons to suitable substrate with regeneration of DMP⁺. Thus DMP⁺ could be used as a catalyst for electroreduction and this possibility was studied. It was found to be a suitable catalyst for the reduction of several difficult-to-reduce substrates (E_1/2 < ?2.8 V vs. SCE). Among others it effected the reduction of fluorobenzene, an aliphatic chloride, and simple conjugated dienes. A possible mechanism and the utility of the reaction are discussed.     

Nickel-catalyzed reductive cleavage of aryl alkyl ethers to arenes in absence of external reductant

The reductive cleavage of the C–O bonds of aryl ethers has great potential in organic synthesis. Although several catalysts that can promote the reductive cleavage of aryl ethers have been reported, all such systems require the use of an external reductant, e.g., hydrosilane or hydrogen. Here, we report the development of a new nickel-based catalytic system that can cleave the C–O bonds of ethers in the absence of an external reductant. The hydrogen atom required in this new reductive cleavage reaction is provided by the alkoxy group of the substrate, which serves as an internal reductant. The absence of an external reductant enables the unique chemoselectivity, i.e., the selective reduction of an alkoxy group over alkenes and ketones.     

Formation and High Reactivity of the anti‐Dioxo Form of High‐Spin μ‐Oxodioxodiiron(IV) as the Active Species That Cleaves Strong C−H Bonds

Recently, it was shown that μ‐oxo‐μ‐peroxodiiron(III) is converted to high‐spin μ‐oxodioxodiiron(IV) through O?O bond scission. Herein, the formation and high reactivity of the anti‐dioxo form of high‐spin μ‐oxodioxodiiron(IV) as the active oxidant are demonstrated on the basis of resonance Raman and electronic‐absorption spectral changes, detailed kinetic studies, DFT calculations, activation parameters, kinetic isotope effects (KIE), and catalytic oxidation of alkanes. Decay of μ‐oxodioxodiiron(IV) was greatly accelerated on addition of substrate. The reactivity order of substrates is toluene<ethylbenzene≈cumene<trans‐β‐methylstyrene. The rate constants increased proportionally to the substrate concentration at low substrate concentration. At high substrate concentration, however, the rate constants converge to the same value regardless of the kind of substrate. This is explained by a two‐step mechanism in which anti‐μ‐oxodioxodiiron(IV) is formed by syn‐to‐anti transformation of the syn‐dioxo form and reacts with substrates as the oxidant. The anti‐dioxo form is 620 times more reactive in the C?H bond cleavage of ethylbenzene than the most reactive diiron system reported so far. The KIE for the reaction with toluene/[D₈]toluene is 95 at ?30 °C, which the largest in diiron systems reported so far. The present diiron complex efficiently catalyzes the oxidation of various alkanes with H₂O₂.     

Enantioselective synthesis of ethyl (S)-2-hydroxy-4-phenylbutyrate by recombinant diketoreductase

Recombinant diketoreductase showed excellent stereoselectivity in the double reduction of β,δ-diketo esters. To investigate the substrate specificity and to broaden the applications of this new biocatalyst, a number of ketone substrates were used to evaluate the substrate spectrum and enantioselectivity of this enzyme in the present study. Among the ketone substrates tested, only this enzyme displayed high efficiency and excellent enantioselectivity in the reduction of ethyl 2-oxo-4-phenylbutyrate to ethyl (S)-2-hydroxy-4-phenylbutyrate. After optimizing the reaction conditions, the bio-reduction of ethyl 2-oxo-4-phenylbutyrate at a substrate concentration of 0.8 M (164.8 g/L) was achieved by the recombinant diketoreductase in an aqueous-toluene biphasic system coupled with formate dehydrogenase for the regeneration of cofactor, resulting in an overall hydroxyl product yield of 88.7% (99.5% ee). This new enzymatic transformation may offer a practical method for the preparation of this important chiral building block.     

On the Catalytic Mechanism of (S)‐2‐Hydroxypropylphosphonic Acid Epoxidase (HppE): A Hybrid DFT Study

The mechanism of oxidative epoxidation catalyzed by HppE, which is the ultimate step in the biosynthesis of fosfomycin, was studied by using hybrid DFT quantum chemistry methods. An active site model used in the computations was based on the available crystal structure for the HppE‐Fe^II‐(S)‐HPP complex and it comprised first‐shell ligands of iron as well as second‐shell polar groups interacting with the substrates. The reaction energy profiles were constructed for three a priori plausible mechanisms proposed in the literature, and it was found that the most likely scenario for the native substrate, that is, (S)‐HPP, involves generation of the reactive Fe^III? O ^. /Fe^IV?O species, which is responsible for the C? H bond‐cleavage. At the subsequent reaction stage, the OH‐rebound, which would lead to a hydroxylated product, is prevented by a fast protonation of the OH ligand and, as a result, ring closure is the energetically preferred step. For the R enantiomer of the substrate ((R)‐HPP), which is oxidized to a keto product, comparable barrier heights were found for the C? H bond activation by both the Fe^III? O₂ ^. and Fe^IV?O species.     

Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry

We investigate the mechanism of disulfide bond cleavage in gaseous peptide and protein ions initiated by a covalently-attached regiospecific acetyl radical using mass spectrometry (MS). Highly selective S–S bond cleavages with some minor C–S bond cleavages are observed by a single step of collisional activation. We show that even multiple disulfide bonds in intact bovine insulin are fragmented in the MS2 stage, releasing the A- and B-chains with a high yield, which has been challenging to achieve by other ion activation methods. Yet, regardless of the previous reaction mechanism studies, it has remained unclear why (1) disulfide bond cleavage is preferred to peptide backbone fragmentation, and why (2) the S–S bond that requires the higher activation energy conjectured in previously suggested mechanisms is more prone to be cleaved than the C–S bond by hydrogen-deficient radicals. To probe the mechanism of these processes, model peptides possessing deuterated β-carbon(s) at the disulfide bond are employed. It is suggested that the favored pathway of S–S bond cleavage is triggered by direct acetyl radical attack at sulfur with concomitant cleavage of the S–S bond (S_H2). The activation energy for this process is substantially lower by ∼9–10 kcal mol^–1 than those of peptide backbone cleavage processes determined by density functional quantum chemical calculations. Minor reaction pathways are initiated by hydrogen abstraction from the α-carbon or the β-carbon of a disulfide, followed by β-cleavages yielding C–S or S–S bond scissions. The current mechanistic findings should be generally applicable to other radical-driven disulfide bond cleavages with different radical species such as the benzyl and methyl pyridyl radicals.