Kinetics Solvent Effect and Mechanism of Hydrolysis of Ethyl Caprylate Ester in Aqueous Organic Solvent Media

The specific rate constant of ethyl caprylate in alkali catalised hydrolysis in water-acetone mixture covering range of 30 to 70% (v/v) of acetone has been determined at temperature 20 to 400c. The rate of reaction decreases with increase in percentage of Acetone from 30 to 70% (v/v). The observed Activation energy decreases progressively with increase in acetone content of the medium. The effect of molar concentration of water and Dielectric constant on the reaction kinetic has also been studied. The thermodynamic parameters (DG*, DH* and DS*) has been determined which showed strong dependency on solvent composition.

Decomposition of Dimethoate and Omethoate in Aqueous Solutions – Half-Life, Neurotoxicity and Mechanism of Hydrolysis

Organophosphate pesticides are used in large quantities. However, they exhibit toxic effects on non-target organisms. Dimethoate and its oxo-analog omethoate inhibit acetylcholinesterase and are toxic for mammals. Moreover, they show extreme toxicity for bees. Once in the environment, they undergo chemical transformations and decomposition. We show that dime-thoate and omethoate decompose rapidly in alkaline aqueous solutions (half-lives 5.7 and 0.89 days) but are highly stable in acidic solutions (half-lives 124 and 104 days). These differences are explained using quantum chemical calculations, indicating that a weaker P–S bond in omethoate is more susceptible to hydrolysis, particularly at a high pH. The toxicity of these pesticides solutions decreases over time, indicating that no or very little highly toxic omethoate is formed during hydrolysis. Presented data can be used to predict dimethoate and omethoate concentrations in contaminated water depending on pH. Presented results suggest that alkaline hydrolysis of organophosphates has an advantage over other techniques for their removal since there is no risk of omethoate accumulation and increased toxicity of contaminated water.

Generalized enzymatic mechanism of catalysis by tetrameric l-asparaginases from mesophilic bacteria

The mechanism of catalysis by the l-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of l-Asn by the type II l-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric l-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (l-Asp and l-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by l-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to l-Asn and l-Gln, we postulate that it is common for all these structurally related enzymes.

Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

The surface treatment of the cardboard in the wet condition by biomodified starch

Поверхностная проклейка бумаги придает ей гидрофобные свойства с одновременным повышением физико-механических и печатных показателей. Крахмал является одним из старейших и наиболее распространенных связующих веществ, используемых в производстве бумаги и картона. Однако в настоящее время вместо нативного крахмала в качестве связующего чаще используются модифицированные крахмалы различного вида. Такие амилолитические ферменты, как амилаза, изоамилаза и пуллуланаза, вызывают энзиматическую модификацию крахмала. Показано, что обработка амилолитическими ферментами крахмала перед поверхностной пропиткой картона заметно улучшает его упругие и деформационные характеристики, поскольку ферментативная модификация крахмала влияет на характер связей, образующихся между крахмалом и волокнами целлюлозы, от чего зависит, какое количество крахмала проникнет между волокнами целлюлозы, а сколько останется на поверхности в виде пленки. От этого соотношения зависит, насколько сильно изменится способность получаемого композитного материала сопротивляться прилагаемой нагрузке. Пропитка картона биомодифицированным крахмалом приводит к увеличению количества водородных связей между волокнами целлюлозы и гидроксильными группами крахмала, что обеспечивает повышенную жесткость при растяжении уже на начальном участке деформирования. При этом повышение физико-механических показателей зависит от вида ферментного препарата и, соответственно, от механизма гидролиза связей в крахмале. Установлено, что обработка картона при 50 сухости амилолитическими ферментами амилазой, изоамилазой или пуллуланазой приводит к значимому увеличению упругости картона после кратковременного увлажнения, но не оказывает значимого положительного эффекта на прочностные характеристики картона при сухости 50 . Paper surface impregnation imparts it hydrophobic properties with a simultaneous increasing in physical-mechanical and printed properties. Starch is one of the oldest and most accepted binders used in the production of paper and cardboard. However, at present time various kinds of modified starch are more often used as a binders instead of native starch. Such amylolytic enzymes as amylase, isoamylase and pullulanase cause enzymatic modification of starch. It has been shown that treated with amylolytic enzymes starch before surface impregnation significantly improves elastic and deformation characteristicsof cardboard, since the enzymatic modification of starch affects the nature of the bonds formed between starch and fibers and determines which part of starch penetrates between the cellulose fibers and which part of starch remains on the surface in the form of a film. It depends the ability of the resulting composite material to resist the applied load. Cardboard impregnation with biomodified starch leads to an increasing in the number of hydrogen bonds between the cellulose fibers and the hydroxyl groups of starch, which provides increased rigidity in tension already at the initial part of the deformation. At the same time, an increasing in physicmechanical parameters depends on the type of enzyme and, accordingly, on the mechanism of hydrolysis of bonds in starch. It has been established that impregnation of cardboard with 50 dryness with amylolytic enzymes amylase, isoamylase or pullulanase leads to a significant increasing in the elasticity of the cardboard after a short-term wetting, but does not have a significant positive effect on the strength characteristics of cardboard with 50 dryness.

Human cholinesterases

The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.

Human cholinesterases

The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.

The Cycle Destruction during Complex Formation of 4-salicylideneamino-3-hydrazino-5-mercapto-1,2,4-triazole with Ni (II) ions

Using single crystal X-ray diffraction method, it was shown that the interaction of 4-salicylideneamino-3-hydrazino-5-mercapto-1,2,4-triazole with Ni (II) acetate yields a complex of nickel with salicylidene-thiosemicarbazone instead of the expected complex with the corresponding Schiff base. A possible mechanism of hydrolysis of the thiotriazole ring is proposed. The N- (salicylidene) thiosemicarbazone is a tridentate and almost flat structure ligand coordinates to the nickel (II) cation through thiosemicarbazone sulphur, phenolic oxygen, and azomethine nitrogen atoms. The nickel (II) ion in the complex is four-coordinated in distorted square-planar geometry. The intermolecular hydrogen bonds N─H∙∙∙N, as well as hydrogen bonds N─H∙∙∙O, O─H∙∙∙O and O─H∙∙∙S through the solvate water molecules, give rise to the 3-D network of the complex [Ni(TSC)((NH2)2CS)]•H2O.

The Catalytic Mechanisms of the Reactions between Tryptophan Indole-Lyase and Nonstandard Substrates: The Role of the Ionic State of the Catalytic Group Accepting the Cα Proton of the Substrate

In the reaction between tryptophan indole-lyase (TIL) and a substrate containing a bad leaving group (L-serine), general acid catalysis is required for the group's elimination. During this stage, the proton originally bound to the C atom of the substrate is transferred to the leaving group, which is eliminated as a water molecule. As a result, the basic group that had accepted the C proton at the previous stage has to be involved in the catalytic stage following the elimination in its basic form. On the other hand, when the substrate contains a good leaving group (-chloro-L-alanine), general acid catalysis is not needed at the elimination stage and cannot be implemented, because there are no functional groups in enzymes whose acidity is strong enough to protonate the elimination of a base as weak as Cl- anion. Consequently, the group that had accepted the C proton does not lose its additional proton during the elimination stage and should take part in the subsequent stage in its acidic (not basic) form. To shed light on the mechanistic consequences of the changes in the ionic state of this group, we have considered the pH dependencies of the main kinetic parameters for the reactions of TIL with L-serine and -chloro-L-alanine and the kinetic isotope effects brought about by replacement of the ordinary water used as a solvent with 2H2O. We have found that in the reaction between TIL and -chloro-L-alanine, the aminoacrylate hydrolysis stage is sensitive to the solvent isotope effect, while in the reaction with L-serine it is not. We have concluded that in the first reaction, the functional group containing an additional proton fulfills a definite catalytic function, whereas in the reaction with L-serine, when the additional proton is absent, the mechanism of hydrolysis of the aminoacrylate intermediate should be fundamentally different. Possible mechanisms were considered.

Substrate-assisted mechanism of catalytic hydrolysis of misaminoacylated tRNA required for protein synthesis fidelity

d-aminoacyl-tRNA-deacylase (DTD) prevents the incorporation of d-amino acids into proteins during translation by hydrolyzing the ester bond between mistakenly attached amino acids and tRNAs. Despite extensive study of this proofreading enzyme, the precise catalytic mechanism remains unknown. Here, a combination of biochemical and computational investigations has enabled the discovery of a new substrate-assisted mechanism of d-Tyr-tRNATyr hydrolysis by Thermus thermophilus DTD. Several functional elements of the substrate, misacylated tRNA, participate in the catalysis. During the hydrolytic reaction, the 2′-OH group of the А76 residue of d-Tyr-tRNATyr forms a hydrogen bond with a carbonyl group of the tyrosine residue, stabilizing the transition-state intermediate. Two water molecules participate in this reaction, attacking and assisting ones, resulting in a significant decrease in the activation energy of the rate-limiting step. The amino group of the d-Tyr aminoacyl moiety is unprotonated and serves as a general base, abstracting the proton from the assisting water molecule and forming a more nucleophilic ester-attacking species. Quantum chemical methodology was used to investigate the mechanism of hydrolysis. The DFT-calculated deacylation reaction is in full agreement with the experimental data. The Gibbs activation energies for the first and second steps were 10.52 and 1.05 kcal/mol, respectively, highlighting that the first step of the hydrolysis process is the rate-limiting step. Several amino acid residues of the enzyme participate in the coordination of the substrate and water molecules. Thus, the present work provides new insights into the proofreading details of misacylated tRNAs and can be extended to other systems important for translation fidelity.