Linear compartmental systems. III. Application to enzymatic reactions

In this paper we extend to enzyme systems the results previously obtained in paper I of this series for linear compartmental systems. We obtain the time course equations for both the enzyme and ligand species involved in the reaction mechanisms, which fit a general enzyme system model when the connections between the different enzyme species are of first or pseudofirst order. The kinetic equations obtained here for a given species, enzyme or ligand have the advantage over all previous equations described in the literature, in that they are in the most simplified form possible, since they only contain the kinetic parameters and initial concentrations of the enzymatic reaction which really have some influence on the time progress curves of the species under study. These kinetic equations are denominated optimized equation to distinguish them from the others, which shall call non-optimized equations. We discuss those cases when both types of equation coincide and we show how, when they do not coincide, the non-optimized equations can be simplified to the optimized ones. Therefore, we show that the optimized equations could be used in all cases to avoid the need of subsequent simplifications to eliminate the parameters that play no role in the corresponding time equations. To illustrate the use of this procedure we will apply it to two simple examples of enzymatic reactions.     

Catalytic enantioselective transamination of alpha-keto esters: an organic approach to enzymatic reactions

The half-transamination reaction of alpha-keto esters with pyridoxamine or 4-picolylamine was found to be catalysed by different metal catalysts in organic solvents giving moderate yields and enantioselectivities of up to 37% ee for methyl-3-indole pyruvate.     

Facile enzymatic aldol reactions with dihydroxyacetone in the presence of arsenate.

Aldol reactions of in situ formed dihydroxyacetone arsenate with different aldehydes were catalyzed by bacterial D-fructose-1,6-bisphosphate aldolase (FruA). Aldolases from bacteria were found to be much more stable and active than FruA from rabbit muscle. Arsenate acts as a phosphate mimic and can, in principle, be used in catalytic amounts. The use of inorganic arsenate and dihydroxyacetone afforded high yields with hydrophobic aldehydes. Cosolvents increased the solubility of hydrophobic aldehydes and afforded higher reaction rates and enzyme stability. Insight is given, for the first time, in the influence of arsenate on the stereoselectivity of the aldol reaction.     

Enantioselective gas chromatographic analysis of aqueous samples by on-line derivatisation. Application to enzymatic reactions

A new gas chromatography (GC) method is presented for analysing both the conversion and the enantiomeric excess (e.e.) of samples from alcohol dehydrogenase reactions. The chiral compounds studied were a series of saturated, straight chain alcohols, ranging from 2-butanol to 2-heptanol. The alcohols were converted to the corresponding trifluoroacetylated derivatives by injecting trifluoroacetic anhydride onto the column shortly after injection of the aqueous samples in split-injection mode (1:100) onto a Chiraldex G-TA capillary GC column. Injecting seven hundred aqueous enzymatic reaction mixtures according to the above-mentioned procedure revealed no noticeable loss of column performance. Using the new GC method, conventional sample work-up procedures such as extraction and off-line derivatisation are eliminated and throughput of samples is significantly enhanced.     

Monosegmented flow-analysis of slow enzymatic reactions: Determination of triglycerides in serum

 A monosegmented flow system (MCFA) is proposed to achieve slow enzymatic spectrophotometric determinations, here applied to the determination of triglycerides in blood serum. The sample (4.5 μL), enzymatic reagent (150 μL) and an air plug (100 μL) are simultaneously inserted into a carrier stream buffered to pH 7.9 (Tris ⋅ HCl). In order to avoid the cumbersome step of air removal, a relocating detector was used. This system handles about 60 samples/h, yielding precise results (r.s.d. usually<2.5%). Sensitivity is 56 mAU ⋅ L/mmol up to 6 mmol/L triglycerides. Accuracy was assessed by running 50 samples already analysed by a conventional procedure yielding the equation C_MCFA(mmol/L)=1.00(±0.04) C_Ref(mmol/L)−0.03(±0.08); r=0.990. Received: 22 January 1997/Revised: 12 March 1997/Accepted: 28 March 1997     

Engineered phenylalanine dehydrogenase in organic solvents: homogeneous and biphasic enzymatic reactions

The use of engineered phenylalanine dehydrogenase N145A supported on Celite for the reductive amination of phenylpyruvic acid in homogeneous and biphasic aqueous-organic solvents is reported. The results indicate that the immobilised biocatalyst is remarkably robust, even in the presence of high concentrations of polar or non-polar organic solvents such as acetone, methanol, n-hexane, toluene and methylene chloride. Cofactor regeneration with alcohol dehydrogenase from Saccharomyces cerevisiae and ethanol was successfully explored. Application to the non-natural poorly water-soluble 2-oxo acid p-NO(2)-phenylpyruvic acid was successfully performed, resulting in the biocatalytic synthesis of p-NO(2)-phenylalanine. In all cases 100% stereoselectivity for the production of the amino acid was retained.     

Valence orbital response to pseudorotation of tetrahydrofuran: a snapshot using dual space analysis

The pseudorotation of tetrahydrofuran (THF) (C(4)H(8)O) has been studied using density functional theory, with respect to the valence orbital responses to the ionization potentials and to orbital electron and momentum distributions. Three conformations of THF, the global minimum structure C(s), local minimum structure C(2), and a transition state structure C(1), which are characteristic configurations on the potential energy surface, are examined using the SAOP/et-pVQZ//B3LYP/6-311++G** models with the aforementioned dual space analysis. It is noted in the ionization energy spectra that the minimum structures C(s) and C(2) are not directly connected by pseudorotation, but through the transition state structure C(1). As a result, some orbitals of the C(s) conformer are able to "correlate" to orbitals of the C(2) conformer without a strict symmetry constraint, i.e., orbital 7a' of the C(s) conformer is correlated to orbital 5b of the C(2) conformer. It is also noted that although the valence orbital ionization potentials are not significantly altered by the pseudorotation of THF, their spectra (mainly due to excitation) are quite different indeed. Detailed orbital analysis based on dual space analysis is given. The valence orbital behavior of the conformations is orbital dependent. It can be approximately divided into three groups: the "signature group" is associated with orbitals experiencing significant changes. The frontier orbitals are in this group. The "nearly identical group" includes orbitals without apparent changes across the conformations. Most of the orbitals showing a certain degree of distortion during the pseudorotation process belong to the third group. The present study demonstrates that a comprehensive understanding of the pseudorotation of THF and its dynamics requires multidimensional information and that the information gained from momentum space is complementary to that from the more familiar coordinate space.     

Weaker Lewis acid,better catalytic activity: dual mechanisms in perfluoroarylborane-catalyzed allylstannation reactions

[reaction: see text] PhB(C(6)F(5))(2) exhibits much higher activity as a Lewis acid catalyst for the allylstannation of aromatic aldehydes than the stronger Lewis acid B(C(6)F(5))(3). This anomalous enhancement of catalytic activity for the weaker LA is shown to be partly due to decreased thermodynamic stability of ion pair 2b relative to 2a in the product-forming step of the reaction. A mechanistic path where the borane serves as the true LA catalyst is more important for the weakly Lewis acidic borane.     

New developments in the field of vitamin B12: enzymatic reactions dependent upon corrins and coenzyme B12.

Simple corrins such as vitamin B₁₂ and vitamin B₁₂ coenzyme catalyze a variety of unusual enzymatic reactions of which some are still without analogy in organic or organometallic chemistry. The mechanisms of these reactions are currently the subject of lively discussion. The present review focuses attention on new ideas about the mode of action of vitamin B₁₂ coenzymes in enzymatic reactions.     

Square-wave protein-film voltammetry: new insights in the enzymatic electrode processes coupled with chemical reactions

Journal of Solid State Electrochemistry - Redox mechanisms in which the redox transformation is coupled to other chemical reactions are of significant interest since they are regarded as relevant...     

Analytical study of two diazotization-coupling reactions. Application to the determination of nano-amounts of nitrite in water

Aluminium has been determined in various standard rocks by 14-MeV neutron activation analysis. The induced ²⁷Mg activity (1.01-MeV photopeak) was counted with a multichannel analyser, equipped with Barto?ek's dead-time stabilizer system. Aluminium standards were prepared by mixing silica and alumina. Correction was made for the interfering reaction ³⁰Si(n,α)²⁷Mg by counting the 1.78-MeV peak, 10 min after irradiation. The mean coefficient of variation on a single determination was 2.8% at the 6–10% aluminium level.     

A new mathematical formula to link near equilibrium relaxation kinetics and conformational selection steps in enzymatic reactions

A new mathematical formula was derived for near equilibrium relaxation processes of enzyme reactions including the conformational selection (CS) modes. CS is one of the most accepted molecular recognition mechanisms, in which protein conformers (CS conformers) are in an equilibrium with varying degrees of ligand binding affinity so that ligands select a particular conformer among them to bind. Using computer simulation techniques, our previous study (Egawa and Callender in Math Biosci 313: 61–70, 2019) predicted that the rate constant for the near equilibrium relaxation processes (k_NER) and the concentration-sum of substrate and product (C_L^t) of enzyme reactions uniquely related to the presence of CS steps in a manner that 1/k_NER versus C_L^t plot transformed from linear to quadratic as the elementary rate constants of inter-conversions among the CS conformers were becoming smaller relative to the rate constants at other steps of the enzymatic reaction system. Thus our previous work could provide a potential tool to detect the presence of CS steps in an enzyme reaction simply by assays using only trace amount of enzyme samples, although logical basis to have the quadratic deformation in the 1/k_NER versus C_L^t plot in response to the presence of CS steps could not be clarified. Employing mathematical approaches that were alternative to those in our previous study, this study succeeded in deriving a theoretical equation that fully explained why and how the CS modes caused the quadratic characters in the 1/k_NER versus C_L^t plot.

     

Highly sensitive and selective pre-column derivatization high-performance liquid chromatography approach for rapid determination of triterpenes oleanolic and ursolic acids and application to Swertia species: optimization of triterpenic acids extraction and pre-column derivatization using response surface methodology

Oleanolic acid (OA) and ursolic acid (UA) are the ubiquitous triterpenic acids in plant kingdom and have multiple biological effects. In the present study, a highly sensitive and selective method using a dual-sensitive probe 2-(5-benzoacridine)ethyl-p-toluenesulfonate (BAETS) as pre-column labeling reagent has been developed for rapid determination of the triterpenes OA and UA by HPLC with fluorescence detection (FLD) and online mass spectrometry identification. Response surface methodology as an efficient tool was employed to optimize the ultrasonic-assisted extraction of triterpenic acids from Swertia plants and the pre-column derivatization reaction, respectively, which ensured the highest triterpenic acids recoveries within the shortest extraction time and the sufficient labeling of the analyzed components. Fast separation of the isomers OA and UA could be achieved on a Hypersil BDS C8 column within 7 min. Both of OA and UA gave the good correlation coefficients of 0.9999. This developed method offered the satisfactory detection limits of 1.10 and 1.30 ng mL⁻¹ for UA and OA, respectively. When applied to Swertia species, it showed good reproducibility.     

Proteins and enzymatic activities in Erbaluce grape berries with different response to the withering process

During the off-vine natural withering process of Erbaluce (white) grapes to obtain “Erbaluce Caluso” Passito wine, some berries change in color from green-yellow to blue. This phenomenon appears at different extents in different years and might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis cinerea loading. To better understand the mechanism involved in color variation, the metabolic changes corresponding to this event were studied.     

A two transition state model for radical-molecule reactions: applications to isomeric branching in the OH-isoprene reaction

A two transition state model is applied to the prediction of the isomeric branching in the addition of hydroxyl radical to isoprene. The outer transition state is treated with phase space theory fitted to long-range transition state theory calculations on an electrostatic potential energy surface. High-level quantum chemical estimates are applied to the treatment of the inner transition state. A one-dimensional master equation based on an analytic reduction from two-dimensions for a particular statistical assumption about the rotational part of the energy transfer kernel is employed in the calculation of the pressure dependence of the addition process. We find that an accurate treatment of the two separate transition state regions, at the energy and angular momentum resolved level, is essential to the prediction of the temperature dependence of the addition rate. The transition from a dominant outer transition state to a dominant inner transition state is shown to occur at about 275 K, with significant effects from both transition states over the 30-500 K temperature range. Modest adjustments in the ab initio predicted inner saddle point energies yield predictions that are in quantitative agreement with the available high-pressure limit experimental observations and qualitative agreement with those in the falloff regime. The theoretically predicted capture rate is reproduced to within 10% by the expression [1.71 x 10(-10)(T/298)(-2.58) exp(-608.6/RT) + 5.47 x 10(-11)(T/298)-1.78 exp(-97.3/RT); with R = 1.987 and T in K] cm3 molecule(-1) s(-1) over the 30-500 K range. A 300 K branching ratio of 0.67:0.02:0.02:0.29 was determined for formation of the four possible OH-isoprene adduct isomers 1, 2, 3, and 4, respectively, and was found to be relatively insensitive to temperature. An Arrhenius activation energy of -0.77 kcal/mol was determined for the high-pressure addition rate constants around 300 K.     

Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection.

The molecular link between the signalling pathway regulating the formation of filopodia and the initiation of local actin polymerization has been elucidated: N-WASP, a close homologue of WASP, which is the product of the gene responsible for the Wiskott-Aldrich syndrome, mediates a direct connection between the small G-protein Cdc42 and the Arp2/3 complex.