Exact and user-friendly kinetic analysis of the two-step rapid equilibrium Michaelis-Menten mechanism

Most enzyme kinetic experiments are carried out under pseudo-first-order conditions, that is, when one of the reactant species (the enzyme or the substrate) is in a large excess of the other species. More accurate kinetic information about the system can be gained without the restrictions of the pseudo-first-order conditions. We present a practical and general method of analysis of the common two-step rapid equilibrium Michaelis-Menten mechanism. The formalism is exact in that it does not involve any other approximations such as the steady-state, limitations on the reactant concentrations or on reaction times. We apply this method to the global analysis of kinetic progress curves for bovine alkaline phosphatase assays carried out under both pseudo-first-order and pseudo-second-order conditions.     

Effective rate models for the analysis of transport-dependent biosensor data.

Optical biosensors, including the BIACORE, provide an increasingly popular method for determining reaction rates of biomolecules. In a flow chamber, with one reactant immobilized on a chip on the sensor surface, a solution containing the other reactant (the analyte) flows through the chamber. The time course of binding of the reactants is monitored. Scientists using the BIACORE to understand biomolecular reactions need to be able to separate intrinsic reaction rates from the effects of transport in the biosensor. For a model to provide a useful basis for such an analysis, it must reflect transport accurately, while remaining simple enough to couple with a routine for estimating reaction rates from BIACORE data. Models have been proposed previously for this purpose, consisting of an ordinary differential equation with 'effective rate coefficients' incorporating reaction and transport parameters. In this paper we investigate both the theoretical basis and numerical accuracy of these and related models.     

Production of biodiesel using a continuous gas-liquid reactor

A novel continuous reactor process has been developed for the production of biodiesel from fats and oils. The key feature of the process is its ability to operate continuously with a high reaction rate, potentially requiring less post reaction cleaning and product/reactant separation than currently established processes. This was achieved by atomising the heated oil/fat and then spraying it into a reaction chamber filled with methanol vapor in a counter current flow arrangement. This allows the continuous separation of product and the excess methanol stream in the reactor. The overall conversion based on a single cycle of this process has been between 50% and 96% of the feed stock materials. Conversions of 94-96% were achieved while operating with 5-7 g of sodium methoxide/L of methanol at methanol flow rate of 17.2 L/h and oil flow rate of 10 L/h. Additional variations in the reactant stoichiometry (i.e. reactant flow rates), catalyst type/concentration, and reaction temperature on the overall product conversion were investigated.     

Construction of a microprocessor-controlled pulsed quench-flow apparatus for the study of fast chemical and biochemical reactions

The construction and operation of a microprocessor-controlled quenched-flow machine are described. Two sets of syringes are moved by high-torque stepping motors to achieve any desired mixing scheme. The dead time of the instrument is pulsed quench-flow operation is of the order of 10 ms. The fastest possible reaction time is 12 ms with the reaction chamber used here; this might be extended downward with other reaction chambers. The reactant volume required is low; 1 ml of solution for each reactant is sufficient for more than 40 kinetic measurements. The machine is tested by alkaline hydrolysis of 2,4-dinitrophenylacetate and used in the investigation of the reaction mechanism of the EcoRI restriction endonuclease and of GTPase activity of ribosomes.     

In situ monitoring of polymerase extension rate and adaptive feedback control of PCR by using fluorescence measurements

Real time PCR detection systems based on fluorescence detection from intercalating dyes (such as SYBR Green I) typically take only single point measurements during every cycle to quantify the amplification. In this process key information about enzymatic kinetics is lost. In this work we measure SYBR Green I fluorescence intensity every 0.5 s within a cycle during PCR in polypropylene tubes. We observe that the intensity during the extension cycle increases while the template is being extended. Results obtained for different lengths are used to estimate an in vitro polymerase activity rate of Thermus aquaticus and Thermus brockianus. An important practical consequence of this result is that the extension time of each PCR cycle can be individually optimized while the reaction is in progress. We demonstrate this idea of adaptive feedback control and show that the total number of cycles and total time required to reach maximum fluorescence is reduced as compared to conventional PCR.     

Methylation in methanol-water mixtures: the effect of solvent composition and high pressure

The effect of pressure (0.1 to 400 MPa) and solvent composition (methanol concentration of 5 to 30%) on the synthesis of beta-methylgalactoside was studied. Galactose was used as a reactant and the reaction was catalyzed by beta-galactosidase from Aspergillus oryzae. Under the applied conditions the enzyme was sufficiently stable and the reaction equilibrium was reached. Higher methanol concentrations obviously influenced the product yield positively due to an increase in reactant concentration but also due to a solvent effect. This solvent effect can be explained by measurement of the activities of galactose and methylgalactoside. These results may be generalized to other methylations in methanol-water systems, where methanol positively affects synthesis yields. Pressure had a small, negative effect on synthesis yields.     

Isoelectric focusing of interacting systems. IV. interaction of macromolecules with each other and with ligands

The theoretical isoelectric focusing behavior for rapidly reversible, bimolecular complexing between two macromolecules depends upon the relative value of the isoelectric point of the complex. When it is intermediate in value, the transient patterns exhibit three peaks. As equilibrium is approached the central peak of complex disappears leaving two reactant peaks. When the isoelectric point is acidic or alkaline to both reactants, the equilibrium pattern also shows two peaks; but in this case only one is pure reactant, the other being a reaction zone. The two cases can be distinguished by varying the relative amounts of reactants. Transient patterns for ligand-binding exhibit a peak of unliganded protein and a reaction zone. As the charged ligand is driven out of the focusing column the reaction zone disappears, so that the equilibrium pattern shows only a peak of unliganded protein. In general, the isoelectric point of the complex cannot be determined from the transient patterns.     

Integrated fragmentation of human IgG and purification of Fab using a reactant adsorptive membrane bioreactor separator system

This article discusses an integrated separation–reaction–separation scheme for producing Fab fragment directly from human immunoglobulin G (hIgG) present in serum feed. The novel reactant adsorptive membrane bioreactor separator (or RAMBS) system used in the current study consisted of a stack of microporous adsorptive membranes held within a temperature controlled module. The membrane stack, in the presence of salt, selectively and reversibly adsorbed hIgG by hydrophobic interaction while allowing most other serum proteins to flow through. The bound hIgG was then fragmented by pumping a solution of papain through the reactor at controlled temperature and flow rate. The salt concentration and pH for reaction and separation were systematically optimized using pure hIgG as reactant. The Fab fragment was separated from undigested hIgG and other byproducts such as Fc fragment based on their differences in hydrophobicity. Under optimal conditions, Fab was obtained in the reaction flow through while the other proteins remained bound to the membrane, these being subsequently eluted by lowering the salt concentration. The RAMBS system in addition to being convenient from process integration and intensification points of view also showed higher catalytic efficiency of papain in comparison to that in liquid phase reactions. Biotechnol. Bioeng. 2009; 104: 152–161 © 2009 Wiley Periodicals, Inc.     

Fermentation data analysis and state estimation in the presence of incomplete mass balance

A method is developed for identifying measurement errors and estimating fermentation states in the presence of unidentified reactant or product. Unlike conventional approaches using elemental balances, this method employs an empirically determined basis, which can tolerate unidentified reaction species. The essence of this approach is derived from the concept of reaction subspace and the technique of singular value decomposition. It is shown that the subspace determined via singular value decomposition of multiple experimental data provides an empirical basis for identifying measurement errors. The same approach is applied to fermentation state estimation. Via the formulation of the reaction subspace, the sensitivity of state estimates to measurement errors is quantified in terms of a dimensionless quantity, maximum error gain (MEG). It is shown that using the empirically determined subspace, one can circumvent the problem of unidentified reaction species, meanwhile reducing the sensitivity of the estimates.     

Incorporation of thiolate donation using 2,2'-dithiodibenzaldehyde: complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

The use of 2,2'-dithiodibenzaldehyde (DTDB) as a reactant for incorporating thiolate donors into the coordination sphere of a transition metal complex without the need for protecting groups is expanded to include the synthesis of complexes with pentadentate ligands. The ligand N,N'-bis(thiosalicylideneimine)-2,2'-thiobis(ethylamine) (tsaltp) is synthesized at a cobalt center by the reaction of DTDB with a Co complex of thiobis(ethylamine). The resulting Co complexes are thus coordinated by the N(2)S(3) pentadentate ligand through two imine N atoms, two thiolate S atoms, and one thioether S atom. A dimeric, bis-thiolate-bridged complex (1) is isolated and converted to a monomeric CN adduct (2) by treatment with KCN. The N(2)S(3) coordination environment provided by the tsaltp ligand is similar to that provided by the protein donors at the active site of the nitrile hydratase enzymes, with 2 being the first octahedral Co complex reported with such a coordination sphere.     

Convection Effects in the BIAcore Dextran Layer: Surface Reaction Model

The BIAcore is a surface plasmon resonance (SPR) device used to measure rate constants, primarily for biochemical reactions. It consists of a flow channel containing one reactant adjoining a dextran gel containing the other. In order to explain anomalous measurements from the device, it has been proposed that some flow penetrates into the dextran layer, thus enhancing transport. A model is presented that accounts for such behavior, and typical velocity fields in the dextran are constructed. The system is analyzed in the limit of the surface reaction model, which corresponds to the limit of thin dextran layers. Asymptotic and singular perturbation techniques are used to analyze association and dissociation kinetics. Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. The results indicate that the effects of such penetration are bound to be small, regardless of the flow model used.     

Phosphorus 31 solid state NMR characterization of oligonucleotides covalently bound to a solid support.

31P cross polarization (CP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra were acquired for various linear and branched di- and tri-nucleotides attached to a controlled pore glass (CPG) solid support. The technique readily distinguishes the oxidation state of the phosphorus atom (phosphate versus phosphate), the presence or absence of a protecting group attached directly to phosphorus (cyanoethyl), and other large changes in the phosphorus chemistry (phosphate versus phosphorothioate). However, differences in configurational details remote from the phosphorus atom, such as the attachment position of the ribose sugar (2'5' versus 3'5'), or the particulars of the nucleotide bases (adenine versus uridine versus thymine), could not be resolved. When different stages of the oligonucleotide synthetic cycle were examined, 31P CPMAS NMR revealed that the cyanoethyl protecting group is removed during the course of chain assembly.     

Fast stopped-flow microcalorimeter

A fast stopped-flow microcalorimeter using a thermistor and an a.c. bridge is described. Time resolution, limited by the response of the thermistor, is 3 ms for t 1/2 while sensitivity is 100 mu degree C in this bandpass. A reaction requires 0.3 ml of each reactant. The microcalorimeter is adiabatic to within 2% for 2 s and mixing may be repeated every 5 min. Computer finite element simulation techniques (FEST) are used to correct for the thermistor response time and heat losses. Results of tests with reactions of NaOH and HCl, NaHCO3, and HCl, and glyclyglycine and CO2 agree with published kinetic and thermodynamic values to within an experimental error of less than 2%.     

Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.     

柑橘属光合作用的环境调节

光合机构的运转受环境影响很大,与柑橘的生长发育、产量和品质密切相关.结合我们的工作,综合论述了柑橘光合作用环境调节的研究进展.强光和紫外光导致光合作用下降与PSⅡ反应中心失活有关,光呼吸和叶黄素循环对光合机构有保护作用.温度胁迫下,光合作用下降主要是RuBPCase活性下降和PSⅡ反应中心失活引起,品种间存在差异.轻度水分胁迫引起的光合作用下降是气孔限制的结果,而严重水分胁迫导致光合作用的非气孔限制.提高CO₂浓度,能够促进柑橘的光合作用,进而促进柑橘的生长和提高其品质.阐述了N、P、S、Fe等矿质元素调节光合作用的机理及盐胁迫对光合作用的影响,指出了今后柑橘光合作用的研究方向.     

Ethanol cycle in an ethanologenic bacterium

A novel redox cycle is suggested, performing interconversion between acetaldehyde and ethanol in aerobically growing ethanologenic bacterium Zymomonas mobilis. It is formed by the two alcohol dehydrogenase (ADH) isoenzymes simultaneously catalyzing opposite reactions. ADH I is catalyzing acetaldehyde reduction. The local reactant ratio at its active site probably is shifted towards ethanol synthesis due to direct channeling of NADH from glycolysis. ADH II is oxidizing ethanol. The net result of the cycle operation is NADH shuttling from glycolysis to the membrane respiratory chain, and ensuring flexible distribution of reducing equivalents between the ADH reaction and respiration.     

Spontaneous Mirror Symmetry Breaking in the Aldol Reaction and its Potential Relevance in Prebiotic Chemistry

The origin of the single chirality of most biomolecules is still a great puzzle. Carbohydrates could form in the formose reaction, which is proposed to be autocatalytic and contains aldol reaction steps. Based on our earlier observation of organoautocatalysis and spontaneous enantioenrichment in absence of deliberate chiral influences in the aldol reaction of acetone and p-nitrobenzaldehyde we suggest that a similar effect might be present also in the aldol reactions involved in gluconeogenesis. Herein we show that reactant precipitation observed in our earlier reported experiments does not affect the asymmetric autocatalysis in the aldol reaction we studied. We explain the phenomenon of spontaneous mirror symmetry breaking in such organocatalytic homogenous systems qualitatively by non-linear reaction network kinetics and classical transition state theory.     

Cooperativity of chirality in homogeneous catalysis: The gold(I)-catalyzed aldol reaction and the vanadium(IV)-catalyzed hetero Diels–Alder cycloaddition

The notion of internal (or intramolecular) cooperativity of chirality is reviewed on the basis of various examples of diastereoisomeric ferrocenylphosphine ligands used in the gold(I)-catalyzed aldol reaction. It was found that the stereochemical outcome of this reaction strongly depends on the specific combination of the absolute configuration of the different stereogenic centers present in the ligand. Thus, individual chirotopic segments in these ligand molecules can act either in a cooperative or noncooperative manner in determining both diastereo-and enantioselectivity. Furthermore, several examples of application of the strategy of double stereodifferentiation (external, or intermolecular cooperativity of chirality) in the gold(I)-catalyzed aldol reaction and the vanadium(IV)-catalyzed hetero Diels–Alder condensation are presented. Based on our work it is apparent that, whether the diastereoselectivity of these two reactions is controlled by the catalyst or by a chiral substrate, cannot be predicted and very much depends on the nature of every individual reactant. Thus, it was found that in both reactions the chiral aldehyde substrate usually has a strong impact on the diastereoselectivity, leading to interesting patterns of double asymmetric induction. On the other hand, chiral isocyanoacetate and chiral-activated dienes, respectively, showed little or no effect on the stereochemical outcome of the reactions.