Influence of the conserved active site residues of histidyl tRNA synthetase on the mechanism of aminoacylation reaction

The relation between the conservation of active site residues and the molecular mechanism of aminoacylation reaction is an unexplored problem. In the present paper, the influences of the conserved active site residues on the reaction mechanism as well as the electrostatic potential near the reaction center are analyzed for Histidyl tRNA synthetase from Escherichia coli, Thermus thermophilus and Staphylococcus aureus. While the primary structures show both convergence as well as divergence, the secondary level structures of the active sites of the three species show considerable conservation in the respective structural organizations. The conserved active site residues near the reaction center, which have a major role in the reaction mechanism and catalysis, retain their specific position and orientation relative to the substrate in the three species. In order to understand the influence of different conserved and nonconserved residues near the reaction center, two different models are considered. First, a large model of active site with the substrates, Mg²⁺ ions and water is constructed in which the first shell residues (including both conserved as well as nonconserved) near the reaction center are studied. From the large model, a smaller model is constructed for reaction path modeling individually for three species. Validation of the smaller model is carried out by comparing the energy surfaces of large and small models as a function of reaction coordinates. Further, the electrostatic potential near the reaction center for the large and small model are compared. The transition state structures of the activation step of aminoacylation reaction for E. coli, T. thermophilus and S. aureus are calculated using the combined ab-initio/semi-empirical calculation. The similarity of the energy profiles as a function of the relevant reaction coordinate and the orientation of the catalytic residue, Arg259, indicate that the reaction mechanisms are identical which are guided by the strikingly similar structural pattern formed by conserved residues for three species. The energy surfaces have close resemblance in three species and present a clear perspective that how the reaction proceeds with the aid of different conserved residues. The study of electrostatic potential confirms this view. The present study provides an understanding of the relationship between the conservation of residues and the efficient reaction mechanism of aminoacylation reaction.     

Sensitivity of the (Na+ + k+)-atpase to state-dependent inhibitors. Effects of digitonin and Triton X-100

Treatment of a purified (NA+ + 5+)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na+ + k+)-atpase reaction inhibited more than the K+-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na+ + k+)-atpase reaction but not the K+-phosphatase reaction; however, treatment with digitonin made the K+-phosphatase reaction almost as sensitive to oligomycin as the (Na+ + k+)-atpase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K+-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K+-phosphatase reaction more than the (Na+ + k+)-atpase reaction). Both digitonin and Triton markedly increased the K0.5 for K+ as activator of the K+-phosphatase reaction, with little effect on the K0.5 for K+ as activator of the (Na+ + k+)-ATpase reaction. In contrast, increasing the K0.5 for K+ in the K+-phosphatase reaction by treatment of the enxyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K+-phosphatase reaction by ATP and increased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K0.5 for K+ in the K+-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na+ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na+ plus nucleotide.     

Kinetic modeling of omega-transamination for enzymatic kinetic resolution of alpha-methylbenzylamine

A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-alpha-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-alpha-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-alpha-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of alpha-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of alpha-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction.     

Acceleration of catalytic activity of calcium oxide for biodiesel production

This research was aimed at studying the acceleration of the catalytic activity of calcium oxide (CaO) for developing an effective heterogeneous catalyst for biodiesel production by the transesterification of plant oil with methanol. CaO was activated by pretreatment with methanol and was used for the transesterification reaction. The activation and transesterification reaction conditions were examined. The obtained optimal reaction conditions were 0.1-g CaO, 3.9-g methanol, 15-g rapeseed oil, and 1.5-h activation time at room temperature that provided methyl ester in approximately 90% yield within a reaction time of 3h at 60 degrees C. The activation mechanism was also investigated, and the proposed mechanism is as follows. By pretreatment with methanol, a small amount of CaO gets converted into Ca(OCH(3))(2) that acts as an initiating reagent for the transesterification reaction and produces glycerin as a by-product. Subsequently, a calcium-glycerin complex, formed due to the reaction of CaO with glycerin, functions as the main catalyst and accelerates the transesterification reaction.     

Facile synthesis of 17-formyl steroids via palladium-catalyzed homogeneous carbonylation reaction

17-formyl-androst-16-ene and its analogues were synthesized from the corresponding 17-iodo-16-ene derivatives in palladium-catalyzed formylation reaction using tributyltin hydride as hydrogen source under mild reaction conditions. The formation of androst-16-ene and its isomerization products, as well as that of analogous steroidal olefins as side-products, was found to be dependent on the reaction conditions. The formylation reaction tolerates various functional groups on the A and B rings of the steroids.     

Studies on the kinetics of reaction and hydrolysis of fluorescamine

The influence of various parameters on the rate of reaction of fluorescamine with primary amines and on the rate of hydrolysis of the reagent is described. The studies indicate that both are dependent on the reaction conditions, including pH, solvent in which the reagent is prepared, temperature, reagent concentration, and buffer salt. Under any set of conditions the reaction rates vary with the amines. A correlation between reaction rate and extent of fluorophor formation has been demonstrated. Kinetic evidence for a multistep reaction mechanism, as well as values for the kinetic constants, are presented.     

Increase in the haemolytic activity of anti-erythrocyte antibodies by anti-gamma-globulin serum

It appears that anti-gamma-globulin sera not only affect the haemagglutinating reaction but can also be employed for detecting antibodies at the erythrocyte surface through the haemolytic reaction. The observed increase in the haemagglutinating as well as haemolytic reaction brought about by anti-gamma-globulin sera is due mainly to the effect on the reaction of 7 S anti-erythrocyte antibodies, the reaction of macroglobulin antibodies being little affected.     

The reaction mechanism of N-benzoylimidazole with ribonucleotides.

The reaction of uridine 3'-phosphate with benzoylimidazole in the absence and presence of a strong base was followed up by 31P and 1H nmr as well as paper electrophoresis. Possible reaction courses were proposed, the reaction rate constants were calculated and the reaction mechanism was discussed. It is possible to selectively acylate ribonucleotides with benzoylimidazole by appropriate choice of the base used.     

Model sclerotization studies. 4. Generation of N-acetylmethionyl catechol adducts during tyrosinase-catalyzed oxidation of catechols in the presence of N-acetylmethionine

Incubation of catechol with mushroom tyrosinase in the presence of N-acetylmethionine resulted in the generation of an adduct. This product was identified to be N-acetylmethionyl catechol, on the basis of spectral characteristics and well-characterized chemical reaction of o-benzoquinone with N-acetylmethionine. Enzyme-catalyzed oxidation of catechol and the subsequent nonenzymatic addition of the resultant quinone to N-acetylmethionine accounted for the observed reaction. That the reaction is not confined to catechol alone, but is of general occurrence, can be demonstrated by the facile generation of similar adducts in incubation mixtures containing N-acetylmethionine, tyrosinase, and different N-acetylmethionines, such as 4-methylcatechol and N-acetyldopamine. Attempts to duplicate the reaction with insect cuticular phenoloxidases were not successful, as the excess N-acetylmethionine used in the reaction inhibited their activity. Nevertheless, occurrence of this nonenzymatic reaction between N-acetylmethionine and mushroom tyrosinase-generated quinones indicates that a similar reaction between enzymatically generated quinones in the cuticle with protein-bound methionine moiety is likely to occur during in vivo quinone tanning as well. Arch. Insect Biochem. Physiol. 38:44–52, 1998. © 1998 Wiley-Liss, Inc.     

Investigation of Embedded Si/C System Exposed to a Hybrid Reaction of Centrifugal-Assisted Thermite Method

A novel method is proposed to study the behavior and phase formation of a Si+C compacted pellet under centrifugal acceleration in a hybrid reaction. Si+C as elemental mixture in the form of a pellet is embedded in a centrifugal tube. The pellet assembly and tube are exposed to the sudden thermal energy of a thermite reaction resulted in a hybrid reaction. The hybrid reaction of thermite and Si+C produced unique phases. X-ray diffraction pattern (XRD) as well as microstructural and elemental analyses are then investigated. XRD pattern showed formation of materials with possible electronic and magnetic properties. The cooling rate and the molten particle viscosity mathematical model of the process are meant to assist in understanding the physical and chemical phenomena took place during and after reaction. The results analysis revealed that up to 85% of materials converted into secondary products as ceramics-matrix composite.     

Visualization of electronic properties of molecules in chemical reactions

Modern computational methods allow for the tracking of entire chemical reactions, ranging from initial reactants, through transition states, and to the final products. They also permit the computation of a variety of properties that can change as the reaction proceeds from start to finish. Visualization of these reactions is often difficult and usually limited to static displays of specific steps along the reaction paths. This article describes a program, Reaction Viewer, that we have developed to visualize a chemical reaction dynamically. The article also describes the use of this program to see the movement of electrons and other electronic effects, as well as steric ramifications during the reaction.     

First Novozym 435 lipase-catalyzed Morita–Baylis–Hillman reaction in the presence of amides

The first Novozym 435 lipase-catalyzed Morita–Baylis–Hillman (MBH) reaction with amides as co-catalyst was realized. Results showed that neither Novozym 435 nor amide can independently catalyze the reaction. This co-catalytic system that used a catalytic amount of Novozym 435 with a corresponding amount of amide was established and optimized. The MBH reaction strongly depended on the structure of aldehyde substrate, amide co-catalyst, and reaction additives. The optimized reaction yield (43.4%) was achieved in the Novozym 435-catalyzed MBH reaction of 2, 4-dinitrobenzaldehyde and cyclohexenone with isonicotinamide as co-catalyst and β-cyclodextrin as additive only in 2 days. Although enantioselectivity of Novozym 435 was not found, the results were still significant because an MBH reaction using lipase as biocatalyst was realized for the first time.     

A Kinetic Analysis of Coupled (or Auxiliary) Enzyme Reactions

As a case study, we consider a coupled (or auxiliary) enzyme assay of two reactions obeying the Michaelis–Menten mechanism. The coupled reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction is the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the coupled reaction is described by a pair of interacting Michaelis–Menten equations. Moreover, we show that when the indicator reaction is fast, the quasi-steady-state dynamics are governed by three fast variables and one slow variable. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis–Menten equations, are derived. The theory can be extended to deal with more complex sequences of enzyme-catalyzed reactions.     

生物结构上的“反应点阵”

细胞膜和各种细胞器膜表面均结合和分布着具有不同生物活性的生物大分子,能引起各种反应,可以看作是一些反应点。进化使这些反应点组合,排列,构成特异的反应点序列,可称为"反应点阵"。     

Measurement of enzyme reaction rates using advanced pH control system: application of repetitive PF system

The aim of this article is to develop the on-line measurement system of enzyme reaction rates, using an advanced pH controller, which is called a repetitive PF(Programmed Controller/Feedback Compensator) System. In the esterolysis reaction of the N-acetyltyrosine ethyl ester by alpha-chymotrypsin, the enzyme reaction rate could be calculated from the amount of base required for keeping the pH constant. The proposed controller has a learning mechanism in which the knowledge is obtained from the former results of the repetition, and pH was controlled much more successfully by the proposed repetitive PF System than by the conventional on-off controller, PI controller, and the adaptive controller. The enzyme reaction rate could be evaluated each time as accurately as possible based on the result of the controlled batch reaction. Using Lineweaver-Burk plots of the estimated reaction rates, kinetic parameters, such as the enzyme activity and Michaelis-Menten constant, could be estimated more accurately and from fewer experiments by the repetitive PF system than by initial reaction rates using on-off pH-stat.     

The effect of agitation intensity on alkali-catalyzed methanolysis of sunflower oil

The sunflower oil methanolysis was studied in a stirred reactor at different agitation speeds. The measurements of drop size, drop size distribution and the conversion degree demonstrate the effects of the agitation speed in both non-reaction (methanol/sunflower oil) and reaction (methanol/KOH/sunflower oil) systems. Drop size distributions were found to become narrower and shift to smaller sizes with increasing agitation speed as well as with the progress of the methanolysis reaction at a constant agitation speed. During the methanolysis reaction, the Sauter-mean drop diameter stays constant in the initial slow reaction region, rapidly decreases during the fast reaction period and finally reaches the equilibrium level. Due to the fact that the interfacial area increases, one can conclude that the rate of reaction occurring at the interface will also be enhanced progressively. The "autocatalytic" behavior of the methanolysis reaction is explained by this "self-enhancement" of the interfacial area, due to intensive drop breakage process.     

海滨锦葵油制备生物柴油工艺条件优化

以海滨锦葵油为原料制备生物柴油。通过单因素试验及正交试验研究了反应温度、催化剂用量、醇油摩尔比、反应时间、搅拌强度等因素对酯交换率的影响。结果表明,在试验范围内各影响因素对酯交换率作用的大小依次为：搅拌强度＞催化剂用量＞醇油摩尔比＞反应时间＞反应温度。海滨锦葵油制备生物柴油的最佳工艺参数为：搅拌强度为1800r.min-1,催化剂KOH用量为海滨锦葵油质量的1%,醇油摩尔比6/1,反应时间60min,反应温度65℃,在该工艺条件下,酯交换反应三次,酯交换率达到97.8%。     

A comparison of the fine-structural localization of nucleoside phosphatase activity in large intracranial blood vessels and the thoracic aorta of rabbits

Summary Nucleoside phosphatase activity was localized in rabbit intracranial blood vessels, namely cerebral and basilar arteries and veins, and in the thoracic aorta using the electron microscope. In the intracranial vessels the same ultrastructural localization of reaction product was found when ATP or ADP was used as substrate in a modified Wachstein-Meisel procedure using Mg ions as the enzyme activator. No reaction product was seen when using AMP or -glycero-phosphate as substrates, or in controls without any substrate. Reaction product was sparsely localized within cell membrane invaginations on all sides of endothelial and, to a lesser extent, of smooth muscle cells. Pinocytotic vesicles occasionally contained reaction product. The greatest amount of lead phosphate (reaction product) precipitate was found in the basement membranes of both endothelial and smooth muscle cells, particularly intense in the former case. A diffuse precipitate of reaction product was observed in the cytoplasm of adventitial fibroblasts.The thoracic aorta demonstrated the same localization with the following exceptions: generally greater concentrations of reaction product were found using ATP as substrate, than in the corresponding intracranial vessels; interendothelial gaps and the cell membrane invaginations of these gaps were completely filled with reaction product; there was no specific localization of reaction product in the basement membranes; and reaction product could also be demonstrated using AMP and glycero-phosphate as substrates. Acid phosphatase activity was localized in lysosomes in endothelial and smooth muscle cells from both types of vessels. The differences in enzyme localization between aorta and intracranial vessels were discussed, particularly in light of the differences in nucleoside phosphatase activity and transport functions between brain and somatic capillaries.     

Tyrosinase catalyzes an unusual oxidative decarboxylation of 3,4-dihydroxymandelate

Tyrosinase usually catalyzes the conversion of monophenols to o-diphenols and oxidation of diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, it catalyzed an unusual oxidative decarboxylation reaction generating 3,4-dihydroxybenzaldehyde as the sole product. The identity of the product was confirmed by high-performance liquid chromatography (HPLC) as well as ultraviolet and infrared spectral studies. None of the following enzymes tested catalyzed the new reaction: galactose oxidase, ceruloplasmin, superoxide dismutase, ascorbate oxidase, dopamine beta-hydroxylase, and peroxidase. Phenol oxidase inhibitors such as phenylthiourea, potassium cyanide, and sodium azide inhibited the reaction drastically, suggesting the participation of the active site copper of the enzyme in the catalysis. Mimosine, a well-known competitive inhibitor of tyrosinase, competitively inhibited the new reaction also. 4-Hydroxymandelic acid and 3-methoxy-4-hydroxymandelic acid neither served as substrates nor inhibited the reaction. Putative intermediates such as 3,4-dihydroxybenzyl alcohol and (3,4-dihydroxybenzoyl)formic acid did not accumulate during the reaction. Oxidation to a quinone methide derivative rather than conventional quinone accounts for this unusual oxidative decarboxylation reaction. Earlier from this laboratory, we reported the conversion of 4-alkylcatechols to quinone methides catalyzed by a cuticular phenol oxidase [Sugumaran, M., & Lipke, H. (1983) FEBS Lett. 155, 65-68]. Present studies demonstrate that mushroom tyrosinase will also catalyze quinone methide production with the same active site copper if a suitable substrate such as 3,4-dihydroxymandelic acid is provided.     

An autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the special characteristics of the enzyme reaction

A moving front has been observed as a special pattern during the hydrogenase-catalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thin-layer reaction chamber. Such fronts start spontaneously and at random times at different points of the reaction chamber; blue spheres are seen expanding at constant speed and amplitude. The number of observable starting points depends on the hydrogenase concentration. Fronts can be initiated by injecting either a small amount of completed reaction mixture or activated hydrogenase, but not by injecting a low concentration of reduced benzyl viologen. These characteristics are consistent with an autocatalytic reaction step in the enzyme reaction. The special characteristics of the hydrogen-uptake reaction in the bulk reaction (a long lag phase, and the enzyme concentration dependence of the lag phase) support the autocatalytic nature. We conclude that there is at least one autocatalytic reaction step in the hydrogenase-catalyzed reaction. The two possible autocatalytic schemes for hydrogenase are prion-type autocatalysis, in which two enzyme forms interact, and product-activation autocatalysis, where a reduced electron acceptor and an inactive enzyme form interact. The experimental results strongly support the occurrence of prion-type autocatalysis.