The multiple Maillard reactions of ribose and deoxyribose sugars and sugar phosphates

Ribose 5-phosphate (R5P) undergoes the Maillard reaction with amines at significantly higher rates than most other sugars and sugar phosphates. The presence of an intramolecular phosphate group, which catalyzes the early stages of the Maillard reaction, provides the opportunity for the R5P molecule to undergo novel reaction paths creating unique Maillard products. The initial set of reactions leading to an Amadori product (phosphorylated) and to an alpha-dicarbonyl phosphate compound follows a typical Maillard reaction sequence, but an observed phosphate hydrolysis accompanying the reaction adds to the complexity of the products formed. The reaction rate for the loss of R5P is partially dependent on the pK(a) of the amine but also is correlated to the protonation of an early intermediate of the reaction sequence. In the presence of oxygen, a carboxymethyl group conjugated to the amine is a major product of the reaction of R5P with N-acetyllysine while little of this product is generated in the absence of oxygen. Despite lacking a critical hydroxyl group necessary for the Maillard reaction, 2-deoxyribose 5-phosphate (dR5P) still generates an Amadori-like product (with a carbonyl on the C-3 carbon) and undergoes phosphate cleavage. Two highly UV-absorbing products of dR5P were amine derivatives of 5-methylene-2-pyrrolone and 2-formylpyrrole. The reaction of dR5P with certain amines generates a set of products that exhibit an interesting absorbance at 340nm and a high fluorescence.     

The kinetics and mechanism of oxidation of reduced spinach ferredoxin by molecular oxygen and its reduced products

Reduced spinach ferredoxin reacts with molecular oxygen in an autocatalytic reaction characterized by a hyperbolic dependence on oxygen concentration. The kinetics of the reaction indicate formation of a reduced ferredoxin-oxygen intermediate complex and production of superoxide anion which may also react with reduced ferredoxin. Hydrogen peroxide, which is formed from superoxide, in turn reoxidizes reduced ferredoxin at a rate nearly 10-times faster than that of the comparable reaction with oxygen. The kinetics of reaction of hydrogen peroxide with reduced ferredoxin are biphasic. The substrate dependence of the first phase of the reaction is consistent with a simple one-step equilibrium reaction. The second phase of the reaction could be eliminated by addition of the radical trapper, sodium formate.     

Reaction time and interhemispheric interaction

The study was made on healthy adult subjects. The reaction time of the hand (RT) was measured under two conditions: 1) the choice of reaction (right or left hand) is determined by the nature of the warning stimulus; 2) decision on the choice is taken, depending on the second, trigger stimulus. Stimuli are presented at random sequences to different visual fields. The reaction time to the visual signal presented to the visual field ipsilateral to the hand is significantly shorter (by 15 to 26 msec) than to the stimulus in the contralateral visual field. In a simple motor reaction, when no discrimination of trigger stimulus and the decision on the choice of reaction is required, a hemispheric asymmetry of reaction time is manifested: the left hemisphere only responds differently to direct visual stimulation and to that mediated through the contralateral hemisphere.     

The reaction between nitrite and deoxyhemoglobin. Reassessment of reaction kinetics and stoichiometry

Recent evidence suggests that the reaction between nitrite and deoxygenated hemoglobin provides a mechanism by which nitric oxide is synthesized in vivo. This reaction has been previously defined to follow second order kinetics, although variable product stoichiometry has been reported. In this study we have re-examined this reaction and found that under fully deoxygenated conditions the product stoichiometry is 1:1 (methemoglobin:nitrosylhemoglobin), and unexpectedly, the kinetics deviate substantially from a simple second order reaction and exhibit a sigmoidal profile. The kinetics of this reaction are consistent with an increase in reaction rate elicited by heme oxidation and iron-nitrosylation. In addition, conditions that favor the "R" conformation show an increased rate over conditions that favor the "T" conformation. The reactivity of nitrite with heme is clearly more complex than has been previously realized and is dependent upon the conformational state of the hemoglobin tetramer, suggesting that the nitrite reductase activity of hemoglobin is under allosteric control.     

Modelling and optimisation of enzymatic separating micro-reactor

A mathematical model of an enzymatic separating microreactor with the electro-osmotic control of reaction component transport rates is analysed. The micro-reactor is considered in a form of a thin channel filled with a gel containing an immobilised enzyme and an adsorbent where the enzyme reaction, the molecular diffusion, the electro-osmotic flux and the adsorption take place. The substrate inhibited enzyme reaction splitting a non-ionic substrate to two non-ionic products is considered. The reactor operates in a periodic regime, when the channel entry is exposed to the periodic substrate concentration pulses. A chromatographic separation of reaction components, therefore, proceeds in the channel. Effects of principal operational parameters of the reactor system—the reaction channel length, the electric current density, the substrate inlet concentration, the rate of adsorption, and the enzyme activity—on resolution of the products at reactor outlet are analysed. The existence of optimum parameter values (maximising the resolution of reaction products) is shown and a multiparametric optimisation of the reactor performance is accomplished.     

Effects of solvent and ionic medium on the kinetics of axial ligand substitution in vitamin B12. Part IV. The reaction between aquocobalamin and the thiocyanate ion in acetonitrile-water mixtures

The rate constants for the reaction of aquocobalamin with the thiocyanate ion were measured as a function of ionic strength and solvent composition in acetonitrile-water mixtures. The reaction is described by a two-step mechanism: the ligation reaction, where the most stable isomer (S-bonded) is formed and the isomerisation reaction (S-bonded to N-bonded thiocyanate). For the ligation reaction a full quantitative analysis of solvent effects could be performed, whereas for the isomerisation reaction only qualitative observations were made. The equilibrium constant for the isomerisation (S-bonded/N-bonded) is large and does not change with the solvent composition. It is found that the transfer Gibbs energies of activation for the ligation reaction are the same as found for the ligand thiourea. The absence of a solvent effect on the isomerisation reaction is a further example of the ability of vitamin B₁₂ to create its own micro environment.     

An improved method for the quantitative determination of hexosamines according to Elson and Morgan

An improved method for performing the Elson-Morgan reaction in the microliter range is described, wherein the proceeding hydrolysis of the sample and the heating of the reaction components is improved. This is accomplished with help of a covered rack. The cover exerts pressure on the plugs of the reaction vials preventing them from bursting open during heating or hydrolysis and preventing water from seeping into the reaction mixture during cooling in a water bath. The reaction vials are readily available from most laboratory suppliers. The simultaneous cooling of all reaction vials is accomplished by immersion of the closed rack in ice-cold water. The rack is also applicable in all reactions, where many samples must be heated and/or cooled at the same time.     

Kinetics and mechanism of peptide synthesis in solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied measuring changes in the fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data couldn't be described by a simple scheme of the second order reaction. The measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: v = kCNaCAEb, where k is the reaction rate constant, CN is the concentration of leucinamide, and LeuNH2, CAE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates. The English version of the paper.     

使用估计的反应自由能预测组成性和可变剪接位点

基因结构预测中的一个重要步骤是精确地识别剪接位点。基于剪接反应的基本物理原则,最大信息原理被应用到剪接反应的理论分析中,进而导出了反应自由能估计表达式。作为一个简化模型,这个表达式能被用来估计一个5′剪接区或者3′剪接区所参与的剪接反应中的自由能变化。它不但较全面地概括了各个碱基之间的关联,而且还考虑了基因组背景概率的影响。这个反应自由能表达式被用来预测了人类基因中的组成性和可变剪接位点,预测结果是令人满意的,其预测能力比得上当前的一些流行方法。这说明最大信息原理可以作为研究某些核酸-蛋白质相互作用系统（如剪接反应）的理论出发点,导出的反应自由能表达式较好地符合了剪接反应过程。     

Oscillatory kinetics of the peroxidase-oxidase reaction in an open system. Experimental and theoretical studies.

1. The oscillations in the peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7)-catalyzed reaction between NADH and O2 are undamped when the reaction is carried out in a system open to both substrates and when 2,4-dichlorophenol and methylene blue are present in the solution. 2. The waveform of the oscillations changes when the concentration of peroxidase is varied. 3. The waveforms obtained experimentally can be simulated by a branched chain reaction model in which the branching is quadratic. 4. A correlation between the present knowledge of the reaction and the model can be made by combining well established and hypothetical reaction steps into a few reaction schemes. A selection among schemes however, is not possible at the present time. 5. Compound III participates in the reaction as an active intermediate. This is possible because dichlorophenol stimulates the break down of compound III.     

Validity of quasi-steady-state and transfer-function representations for input-output relation in a Michaelis-Menten reaction

In relation to the input-output characteristics of enzymatic reactions in the cellular metabolism and biochemical reactors, the validity of the quasi-steady-state and transfer-function representations of reaction velocity has been examined for a basic Michaelis-Menten reaction employing computer simulation, that is, numerical integration of the rate equation. The well-known S-v relationship (relationship between substrate concentration and reaction velocity)derived on the quasi-steady-state assumption is found to be in general a good approximation to the actual velocity throughout the temporal progress of the reaction. The validity of the approximation depends on a ratio of the Michaelis constant to the total enzyme concentration in the reaction system rather than on the individual rate constants. A transfer-function representation is derived on assuming an exponential change in the reaction velocity for the indicial response to the substrate influx rate. The representation has a wider valid region with a decrease in influx rate than with an increase in the influx rate. The validity is most dependent on a ratio of total enzyme concentration to the steady-state concentration of the substrate. The analysis of the linear sensitivity of the reaction velocity to rate constants reveals that the characteristics of these valid representations in systems analysis change according to the phase of the reaction.     

Use and abuse of the quasi-steady-state approximation

The transient kinetic behaviour of an open single enzyme, single substrate reaction is examined. The reaction follows the Van Slyke-Cullen mechanism, a spacial case of the Michaelis-Menten reaction. The analysis is performed both with and without applying the quasi-steady-state approximation. The analysis of the full system shows conditions for biochemical pathway coupling, which yield sustained oscillatory behaviour in the enzyme reaction. The reduced model does not demonstrate this behaviour. The results have important implications in the analysis of open biochemical reactions and the modelling of metabolic systems.     

Theoretical calculations on hydrogenase kinetics: explanation of the lag phase and the enzyme concentration dependence of the activity of hydrogenase uptake

Two models of the hydrogenase reaction cycle were investigated by means of theoretical calculations and model simulations. The first model is the widely accepted triangular hydrogenase reaction cycle with minor modifications; the second is a modified triangular model, where we have introduced an autocatalytic step into the reaction cycle. Both models include a one-step activation reaction. The theoretical calculations and model simulations corroborate the assumed autocatalytic reaction step concluded from the experimental characteristics of the hydrogenase reaction.     

Enzymatic formation and esterification of (S)-mandelonitrile

The (S)-selective hydroxynitrile lyase from Hevea brasiliensis (HbHNL) catalyzes the trans-cyanohydrin reaction (transcyanation). The equilibrium of this two-step reaction sequence is not favorable unless a large excess of acetone cyanohydrin (1) is used. Therefore, the coupling of this reaction with a follow-up reaction was investigated. It was established that the trans-cyanohydrin reaction could be performed in organic media, making it possible to couple it with a lipase-catalyzed acylation. Candida antarctica lipase B (CAL-B) shows a high selectivity (E=100) for (S)-mandelonitrile (4) and is, therefore, the ideal candidate for this type of multi-step one-pot reaction.     

The peroxidase-catalysed oxidation of a chalcone and its possible physiological significance

1. Crystalline horseradish peroxidase catalysed the oxidation of 2',4,4'-trihydroxychalcone (isoliquiritigenin) in the presence of trace amounts of hydrogen peroxide under aerobic conditions. One atom of oxygen was consumed for each molecule of substrate. 2. The reaction course comprised a lag phase and a linear phase. The optimum pH for the linear phase of the reaction was about 7.5. The length of the lag phase decreased with increasing pH. It is suggested that the chalcone anion is the actual substrate for the reaction. 3. No evidence for the production of reducing free radicals or perhydroxyl radicals during the reaction could be found. 4. 4',7-Dihydroxyflavonol and 4',6-dihydroxyaurone were isolated from the reaction mixture. The immediate products of the reaction may have included 3,4',7-trihydroxyflavanone and 4',6-dihydroxy-2-(alpha-hydroxybenzyl)coumaran-one, which can be readily converted non-enzymically into the flavonol and aurone respectively. 5. A similar reaction was catalysed by cell-free extracts of hypocotyls of Phaseolus vulgaris. 6. The physiological significance of the reaction is discussed in terms of a possible free-radical mechanism. An analogy may exist between flavonoid biosynthesis and lignin formation.     

Type I photosynthetic reaction centres: structure and function

We review recent advances in the study of the photosystem I reaction centre, following the determination of a spectacular 2.5 A resolution crystal structure for this complex of Synechococcus elongatus. Photosystem I is proving different to type II reaction centres in structure and organization, and the mechanism of transmembrane electron transfer, and is providing insights into the control of function in reaction centres that operate at very low redox potentials. The photosystem I complex of oxygenic organisms has a counterpart in non-oxygenic bacteria, the strictly anaerobic phototrophic green sulphur bacteria and heliobacteria. The most distinctive feature of these type I reaction centres is that they contain two copies of a large core polypeptide (i.e. a homodimer), rather than a heterodimeric arrangement of two related, but different, polypeptides as in the photosystem I complex. To compare the structural organization of the two forms of type I reaction centre, we have modelled the structure of the central region of the reaction centre from green sulphur bacteria, using sequence alignments and the structural coordinates of the S. elongatus Photosystem I complex. The outcome of these modelling studies is described, concentrating on regions of the type I reaction centre where important structure-function relationships have been demonstrated or inferred.     

Hybrids of sugars and aromatics: A Pd-catalyzed modular approach to chromans and isochromans

Herein we describe the synthesis of highly substituted chromans and isochromans using carbohydrates as starting materials. The key step of our synthetic approach is the annelation of the benzene moiety via a highly efficient Pd-catalyzed domino reaction. This powerful approach led to a small library of highly substituted chromans and isochromans by making use of a variety of different diynes and bromoglycals. We investigated several Pd-catalysts in order to improve the yields and to enlarge the scope of the domino reaction. Furthermore, we elucidated the mechanistic picture of the reaction with isotope-labelling experiments. Most probably the reaction proceeds via an oxidative addition followed by two carbopalladation steps and a final cyclization reaction.     

Spectral and kinetic studies on the interaction between oxyhaemoglobin and peroxynitrite: a comparison with hydrogen peroxide.

Interaction between oxyhaemoglobin and peroxynitrite was studied using stopped-flow rapid-scan spectrophotometry. The influence of pH, peroxynitrite concentration and temperature on the pseudo-first-order rate constants was studied and the activation energy calculated. The kinetic curve for the oxyhaemoglobin-peroxynitrite reaction showed that a fast reaction occurred in the initial seconds, followed by a slow process of decrease in absorbance. The biphasic reaction kinetics of oxyhaemoglobin with peroxynitrite or hydrogen peroxide demonstrated the existence of an intermediary species. For the first time a rapid-scan stopped-flow spectrophotometry study is presented, yielding spectral and kinetic data of the reaction.     

Kinetic study on the reaction mechanism of pantothenase: existence of an acyl-enzyme intermediate and role of general acid catalysis.

A kinetic study was performed on the reaction mechanism of pantothenase (EC 3.5.1.22) catalyzed hydrolysis of the pantothenic acid. A nonlinear progress curve is derived if the reaction occurs at low buffer concentrations. The nonlinearity is due to partial reversibility of the reaction; an acylenzyme (pantoyl-enzyme) is formed during the reaction, and beta-alanine, the other end product, is able to react with the acyl-enzyme and return back to pantothenate. The dependence of the beta-alanine return reaction on buffer concentration and on pH suggests a general acid catalysis during the reaction. A reaction mechanism is suggested, in which the -NH3+ form of beta-alanine participates in the return reaction, and the deacylation of the acyl-enzyme is acid catalyzed.     

Flavin-photosensitized reactions of retinal and stilbene

1. Retinol and stilbene are both isomerized when they are illuminated anaerobically in the presence of flavins. 2. Triplet quenchers (e.g. oxygen, potassium iodide and paramagnetic ions) inhibit the reaction more efficiently than they quench flavin fluorescence. At 77 degrees C in a diethyl ether-isopentane-ethanol (5:5:2) glass retinol quenches flavin phosphorescence, but not its fluorescence. 3. For the stilbene reaction cis/trans photostationary-state mixtures are obtained with different flavins and these are linearly related to the phosphorescence transition energies of the flavins used. 4. The reaction involves the triplet state of flavin and a scheme for the reaction is suggested. 5. The dependence of the rate of reaction on substrate concentration is explicable in terms of this scheme. 6. The photobleaching of rhodopsin sensitized by flavin is also demonstrated.