Distinguishing reversible from irreversible virus capsid assembly

Capsids of spherical viruses may be constructed from hundreds or thousands of copies of the major capsid protein(s). These assembly reactions are poorly understood. Here we consider the predicted behavior for assembly where the component reactions have weak association energy and are reversible and compare them to essentially irreversible reactions. The comparisons are based on mass action calculations and the behavior predicted from kinetic simulations where assembly is described as a cascade of low order reactions. Reversible reactions are characterized by a pseudo-critical concentration, whereas irreversible reactions consume all free subunits. Irreversible reactions are more susceptible to kinetic traps comprised of numerous small intermediates. In the case where only the ultimate step is irreversible, very low concentrations of intermediates slow the completion of the reaction so that overall it closely matches the predictions for the reversible reactions that make up the majority of the cascade. Data in the literature strongly support the hypothesis that most viruses are held together by many weak interactions.     

Identifying and understanding drug allergies

Drug hypersensitivity reactions frequently occur in hospitalized and out-patients. Clinical presentations are numerous and heterogeneous, from a mild urticaria to a dramatic anaphylactic shock and an extensive bullous skin disease. Allergic reactions are unpredictable reactions, related to immunologic mechanisms. Some reactions mimic allergic reactions but no drug specific antibody or T cell proliferation can be demonstrated. A true diagnosis is rarely set up and the tools for it are lacking. In this review, we will focus on the available epidemiological data concerning these reactions, including data on incidence and mortality and on the most recent advances in the pathophysiology and allergy diagnosis of drug hypersensitivity reactions.     

Catalytic studies on tryptophanase from Bacillus alvei

Tryptophanase from Bacillus alvei exhibited the expected spectrum of pyridoxal-5'-phosphate-dependent reactions. It exhibited l-serine dehydratase, S-alkyl-cysteine lyase, and cysteine desulfhydrase activities, as well as the classic tryptophanase reactions (all beta elimination reactions). It also acted as a tryptophan synthetase (beta replacement reactions) using indole plus l-serine or l-cysteine or S-methyl-cysteine as substrates. The beta elimination reactions are simple competitors of the replacement reactions for the same amino acid substrates. The kinetics of the reactions were examined in detail using a coupled continuous spectrophotometric assay. A product (indole) inhibition study of the beta elimination reaction with tryptophan showed simple, noncompetitive inhibition; the same study with allosubstrates showed noncompetitive inhibition by indole. These product studies provided data on the beta replacement reactions as well. The results are discussed in terms of a mechanism for the B. alvei tryptophanase.     

Constraints and missing reactions in the urea cycle.

The stoichiometric relations in a series of biochemical reactions are summarized by a stoichiometric number matrix (with a column for each reaction) and a conservation matrix (with a row for each constraint). These two matrices for a series or cycle of biochemical reactions are related because the columns of the stoichiometric number matrix are in the null space of the conservation matrix, and the rows of the transpose of the conservation matrix are in the null space of the transpose of the stoichiometric number matrix. The conservation matrix for a system of biochemical reactions is of interest because it shows the nature of the constraints in addition to the conservation of atoms and groups. Constraints beyond those for the conservation of atoms and groups indicate "missing reactions" that do not occur because the enzymes involved couple reactions that could occur and still conserve atoms and groups. The interpretation of conservation matrices and stoichiometric matrices for a reaction system is complicated by the fact that they are not unique. However, their row-reduced forms are unique, as are their dimensions, which represent the number of reactants and number of independent reactions. Two matrices that look different contain the same information if they have the same row-reduced form. The urea cycle, which involves five enzyme-catalyzed reactions, and its net reaction are discussed in terms of the linear constraints produced by enzyme catalysis. A procedure to obtain a set of conservation equations that will yield the correct net reaction is described.     

Thermodynamic yield predictions for biodegradation through oxygenase activation reactions

Activation reactions involve modification of recalcitrant substrates to forms that are more readily degradable. These reactions require specialized enzymes and cosubstrates, including molecular oxygen and reduced electron carriers. In these reactions, microorganisms invest electrons and cannot capture energy or carbon for synthesis. The subsequent degradation of the intermediates formed in activation reactions releases electrons, energy, and carbon that the organisms use for growth. The overall yield is reduced due to the required activation investments. A mathematical method to predict cell yields of oxygenase activation reactions is developed using electron and energy balances. Predicted yields are compared with experimental yields for methane, organic chelating agents, and aromatic hydrocarbons.     

Allergic delayed skin reactions from lipid fractions of trichophytin.

Crude trichophytin was fractionated to find out if its lipid fraction could cause inflammatory delayed allergic skin reactions in dermatophyte-sensitized guinea pigs. The following fractions were obtained: polysaccharide-peptide, total lipids, total lipids without free fatty acids and free fatty acids. The crude trichophytin and polysaccharide-peptide fraction gave rise to strong and equal allergic delayed skin reactions after 24 h. The total lipids gave statistically significant weaker, but clearly positive reactions, and of the same degree as the free fatty acid fraction. The total lipids without free fatty acids did not produce reactions in the sensitized animals, indicating that free fatty acids are responsible for the allergic skin reactions. In some cases the free fatty acids showed comparatively intense reactions. It can be concluded that free fatty acids are antigenic substances that are, sometimes, involved in the allergic delayed skin reactions in dermatophytosis.     

Drug metabolism in the newborn

The duration and intensity of drug action depend not only on the dose of the drug but also on the rates at which drugs are transformed to products that can be excreted readily by the kidney. Two general categories of drug metabolism occur in the liver: phase 1 reactions (oxidations-reductions and hydrolyses) and phase 2 reactions (synthetic conjugations). Phase 1 reactions produce functional groups that can participate in phase 2 reactions. Phase 1 reactions are almost nonexistent in the fetuses of laboratory animals; however, many appear in primates during the first trimester of gestation. Phase 2 reactions are deficient prenatally in both rodents and primates. Parturition triggers a surge of both phase 1 and phase 2 reactions. The lack of uniformity of the development of phase 1 oxidative reactions during the early neonatal period reflects the multiplicity of cytochrome P-450 hemoproteins, the terminal oxidases responsible for most hepatic oxidative biotransformations. The rate of recovery of chemically induced losses of cytochrome P-450 systems is age dependent.     

Modeling speciation effects on biodegradation in mixed metal/chelate systems

A new model, CCBATCH, comprehensively couples microbially catalyzed reactions to aqueous geochemistry. The effect of aqueous speciation on biodegradation reactions and the effect of biological reactions on the concentration of chemical species (e.g. H₂CO₃, NH ₄ ⁺ , O₂) are explicitly included in CCBATCH, allowing systematic investigation of kinetically controlled biological reactions. Bulk-phase chemical speciation reactions including acid/base and complexation are modeled as thermodynamically controlled, while biological reactions are modeled as kinetically controlled. A dual-Monod kinetic formulation for biological degradation reactions is coupled with stoichiometry for the degradation reaction to predict the rate of change of all biological and chemical species affected by the biological reactions. The capability of CCBATCH to capture pH and speciation effects on biological reactions is demonstrated by a series of modeling examples for the citrate/Fe(III) system. pH controls the concentration of potentially biologically available forms of citrate. When the percentage of the degradable substrate is low due to complexation or acid/base speciation, degradation rates may be slow despite high concentrations of substrate Complexation reactions that sequester substratein non-degradable forms may prevent degradation or stopdegradation reactions prior to complete substrate utilization. The capability of CCBATCH to couple aqueous speciation changes to biodegradation reaction kinetics and stoichiometry allows prediction of these key behaviors in mixed metal/chelate systems.     

Effect of pressure on electron transfer reactions in inorganic and bioinorganic chemistry

Kinetic and thermodynamic studies involving the application of different high-pressure techniques, are very useful in gaining mechanistic information on the basis of volume changes that occur during inorganic and bioinorganic electron transfer reactions. The most fundamental type of electron transfer reaction concerns self-exchange reactions, for which the overall reaction volume is zero, and activation volumes can be measured and discussed. In the case of non-symmetrical electron transfer reactions, intra- and intermolecular processes can be studied and volume profiles can be constructed. Precursor complex formation can in some cases be recognized kinetically in such systems. Typical values of activation and reaction volumes are reviewed for various reversible and irreversible electron transfer reactions. Mechanistic conclusions reached on the basis of these parameters are presented. Volume profiles for electron transfer reactions enable a simplistic presentation of the reaction mechanism on the basis of intrinsic and solvational volume changes along the reaction coordinate.     

Tools and ingredients for the biocatalytic synthesis of metabolites

Metabolic networks have been an interesting starting point not only for the design of synthetic routes in a similar sequence of reactions, e.g., in biomimetic syntheses, but also for assembling a number of biocatalytic steps by preparing the required enzymes and auxiliary reagents. Retrosynthetic analysis involving multiple biocatalytic reactions steps therefore needs to consider the practically realized biocatalytic single steps. The opportunities for route selection are enlarged if novel synthetic reactions connecting easily available starting materials and products are found, and/or both biocatalytic and classical reactions of organic chemistry are utilized. Tools and ingredients for biocatalytic synthesis are of special interest for reactions difficult to achieve by classical organic synthesis. Densely and differentially functionalized small molecules do not allow much space for protecting or activating groups. Biocatalytic reactions have therefore performed well for a number of useful metabolites in enantiopure form to achieve full functionality. Although many well-known metabolites from classical biochemistry have only been prepared in racemic form, it is of fundamental interest to have these available in enantiomerically pure form. Biocatalytic reactions with nature's privileged chiral catalysts appear to be a promising synthetic strategy towards these metabolites, especially when sensitive or stable-isotope-labeled metabolites are to be prepared. The main applications for these metabolites are as references materials in metabolomics, as enzyme substrates for the characterization of metabolic enzyme activities and as potential pharmaceuticals in biomedical research. The use of stable-isotope-labeled metabolites can thereby simplify in vivo applications and metabolic flux analyses.     

Secondary kinase reactions catalyzed by yeast pyruvate kinase.

1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.     

Theoretical and practical aspects of glutaraldehyde fixation

Synopsis This review first considers the many structures put forward for glutaraldehyde, and the purification of the commercial material for chemical, histological and histochemical studies. Some practical and theoretical problems of tissue fixation with glutaraldehyde, including artefacts, are then discussed. The chemical reactions with amino acids and proteins are considered next together with the physical changes in the proteins during the reactions. The known reactions of glutaraldehyde with nucleic acids, lipids and mucosubstances are explored briefly.     

Bioorganic reactions in microemulsions: the case of lipases

Water-in-oil microemulsions, or reverse micelles, are being evaluated as a reaction medium for a variety of enzymatic reactions. These systems have many potential biotechnological applications. Important examples are the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. This review illustrates the biotechnological applications of microemulsions as media for bioorganic reactions. The principal focus is on lipase catalyzed processes.     

The temperature dependences of some types of gaseous ionic reactions of astrochemical interest.

The rate constants of ion-molecule reactions which are of potential significance in astrochemical systems are found to exhibit significant, and in many cases, negative temperature dependences. The rate constants of fast ion-polar molecule reactions (e.g., XH+ + B leads to BH+ + X) may increase by a factor of 5-10 between 1000 and 10D. Slow reactions that proceed via reaction complexes (e.g., H- transfer and association reactions) often exhibit temperature dependences of the form k = AT-n, n = 1-5. Both transition state theory considerations and the coupled-oscillator RRK-type model are seen to be able to account qualitatively for the behavior of slow ion-molecule reactions.     

Cantilever Arrays for Multiplexed Mechanical Analysis of Biomolecular Reactions

Microchips;ontaining arrays of cantilever beams have been used to mechanically detect and quantitatively analyze multiple reactions of DNA hybridization and antigen-antibody binding simultaneously. The reaction-induced deflection of a cantilever beam reflects the interplay between strain energy increase of the beam and the free energy reduction of a reaction, providing an ideal tool for investigating the connection between mechanics and chemistry of biomolecular reactions. Since free energy reduction is common for all reactions, the cantilever array forms a universal platform for label-free detection of various specific biomolecular reactions. A few such reactions and their implications in biology and biotechnology are discussed.     

Phototoxic and photoallergic skin reactions

Indirect action of sun together with different exogenous agents (systemic medications and topically applied compounds) sometimes may result in phototoxicic and photoallergic reactions. Drug-induced photosensitivity reactions refer to the development of cutaneous disease as a result of the combined effects of a drug and light (mostly spectrum within the UVA and visible light range or UVB range). The aim of the review was to show the prominent features of phototoxic and photoallergic reactions, which occur in sun-exposed areas, including face, neck, hands and forearms. Phototoxic reactions are significantly more common than photoallergic reactions and mostly resemble to exaggerated sunburn. Photoallergic reactions appear only in a minority of individuals and resemble allergic contact dermatitis on sun-exposed areas, although sometimes may extend into covered areas. Generally, the physical examination and a positive patient's history of photosensitivity reactions on substances are of great importance for the diagnostics. The treatment of these reactions includes identification and avoidance of offending agent and application of anti-inflammatory dressings, ointments and corticosteroids.     

MRAD: Metabolic reaction analysis database--an entity-relationship approach

The Metabolic Reaction Analysis Database (MRAD) is a relational database based on the Entity-Relationship (ER) model which combines information about organisms, biochemical pathways, reactions, enzymes, substrates, products and genes. It describes 244,596 genes in 79 organisms, 6,552 enzymes, and 3,552 reactions, 3,100 substrates, 2,866 products and 118 metabolic pathways. The MRAD graphical user interface allows for the identification of metabolic reactions which are similar and dissimilar in multiple organisms, reactions in a pathway which are missing in an organism and using any combination between one to six of the biological entities of organisms, genes, pathways, enzymes, substrates and products to determine metabolic reactions. MRAD provides a powerful and efficient tool for the construction of flux balance models for metabolic engineering applications.     

Autoxidation reactions of hemoglobin A free from other red cell components: a minimal mechanism

Rates of autoxidation reactions are determined for normal human hemoglobin A preparations which are extensively purified to remove all other redox active red cell components. The effects of superoxide dismutase, catalase, and hydroxyl radical scavengers on the reaction provide evidence for superoxide formation as the rate determing step followed by fast reactions that involve peroxide and hydroxyl radical. These results support a minimum overall mechanism for heme iron(II) oxidation and dioxygen reduction to water. Side reactions also occur that result in the modification and precipitation of the protein moiety; catalase and hydroxyl radical scavengers reduce the extent of the side reactions. These studies provide insight into the basis of oxidant stress in the red cell.     

Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in 'Birch-like' reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., -1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.     

The CanOE strategy: integrating genomic and metabolic contexts across multiple prokaryote genomes to find candidate genes for orphan enzymes

Of all biochemically characterized metabolic reactions formalized by the IUBMB, over one out of four have yet to be associated with a nucleic or protein sequence, i.e. are sequence-orphan enzymatic activities. Few bioinformatics annotation tools are able to propose candidate genes for such activities by exploiting context-dependent rather than sequence-dependent data, and none are readily accessible and propose result integration across multiple genomes. Here, we present CanOE (Candidate genes for Orphan Enzymes), a four-step bioinformatics strategy that proposes ranked candidate genes for sequence-orphan enzymatic activities (or orphan enzymes for short). The first step locates "genomic metabolons", i.e. groups of co-localized genes coding proteins catalyzing reactions linked by shared metabolites, in one genome at a time. These metabolons can be particularly helpful for aiding bioanalysts to visualize relevant metabolic data. In the second step, they are used to generate candidate associations between un-annotated genes and gene-less reactions. The third step integrates these gene-reaction associations over several genomes using gene families, and summarizes the strength of family-reaction associations by several scores. In the final step, these scores are used to rank members of gene families which are proposed for metabolic reactions. These associations are of particular interest when the metabolic reaction is a sequence-orphan enzymatic activity. Our strategy found over 60,000 genomic metabolons in more than 1,000 prokaryote organisms from the MicroScope platform, generating candidate genes for many metabolic reactions, of which more than 70 distinct orphan reactions. A computational validation of the approach is discussed. Finally, we present a case study on the anaerobic allantoin degradation pathway in Escherichia coli K-12.