Long-term continuous reaction of immobilized β-fructofuranosidase

Summary -Fructofuranosidase was immobilized by alginate gel at high efficiency (92 %). The extreme long-term continuous reaction (half-life, 275 days) was achieved by the immobilized enzyme using sucrose at high concentration (500 mg ml^–1) to produce fructo-olicosaccharides, such as 1-kestose (Fru21Fru21aGlc) and nystose (Fru21Fru21Fru21aGlc).     

Revisitation of the βCl-elimination reaction of D-amino acid oxidase: new interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms

D-amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl(-) from βCl-D-alanine via abstraction of the substrate α-H as H(+) ("carbanion mechanism") (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-βCl-D-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈50 s(-1). This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail.     

Calorimetric studies of the haemoglobin–haptoglobin reaction

Haptoglobin binds haemoglobin so firmly that there is practically no dissociation. It would be expected that the heat of the reaction would be relatively large. The development of the microcalorimeter by Benzinger offered the opportunity to measure the heat of reaction. The experiments were carried out in the Beckman 190B Microcalorimeter in two ways: (1) a constant amount of haptoglobin (Kabi; 65mg.) with different amounts of haemoglobin, and (2) a constant amount of haemoglobin (32.5mg.) with different amounts of haptoglobin. The proteins, each in 5ml. of 0.15m-phosphate buffer, pH7.4, were placed in equal-volume calorimeter cells. The heat produced/mg. of haemoglobin was calculated from the slope of the curve for a constant amount of haptoglobin and from the maximum heat for a constant amount of haemoglobin. This heat is about 70kcal./mole at 37 degrees . DeltaH varies with temperature, being -70.2 at 37 degrees , -29.7 at 20 degrees and 7.2 at 4 degrees . From the amount of haptoglobin required to attain maximum heat with 32.5mg. of haemoglobin and the amount of haemoglobin required to attain maximum heat with 65mg. of haptoglobin, it appears that at excess of haptoglobin there is competition between the reactions of 2moles of haptoglobin with 1mole of haemoglobin (or 2 alphabeta-chains) and 1mole of haptoglobin with 1mole of haemoglobin.     

Synthesis of mannose oligosaccharides via reversal of the α-mannosidase reaction

Summary Three naturally occurring isomers of the disaccharideO--d-mannosyl-d-mannoside were synthesized by reversing the hydrolytic activity of jack bean -mannosidase at 75°C in a very high concentration of mannose. Higher oligosaccharides were also obtained at the later stages of the reaction. The maximum total yield of disaccharides was 37% (w/w) based on the total amount of saccharides.     

Oxidase–peroxidase reaction: kinetics of peroxidase-catalysed oxidation of 2-aminophenol

Possible reaction pathways that may lead to horseradish peroxidase inactivation during the aerobic oxidation of 2-aminophenol were investigated using extended kinetic curves. A kinetic model involving the formation of a low-reactive species, Compound III, was proposed and several rate constants were calculated using an optimisation computing program. Sensitivity analysis allowed to conclude that both oxidase and peroxidase cycles occur in 2-aminophenol oxidation.     

Reverse K?nigs-Knorr reaction. Synthesis of beta-D-gluco-thio-cerebroside

The synthesis of 1-thio-beta-D-glucocerebroside by reaction of 1-iodo-3-O-benzoylceramide with 1-mercapto-beta-D-glucopyranose in the presence of sterically hindered amine (DBU) is described.     

Time dependent behaviour of the cross-linking reaction of α-chymotrypsin with glutaraldehyde

Summary Immobilization of -chymotrypsin was done by cross-linking the enzyme with glutaraldehyde. Studies were done in a batch reactor to find out the effects of the concentrations of -chymotrypsin and glutaraldehyde on the rate of particle formation, the final yield of the crosslinked particles, and the residual activity of the cross-linked enzyme. Both intra- and inter-molecular cross-linking play important role at different time course of the reaction. At the begining of the cross-linking reaction, the large drop in the activity is because of the intra-molecular cross-linking and later on the large increase in the particle formation is because of the inter-molecular cross-linking.     

The mechanism of the periodate–thiobarbituric acid reaction of sialic acids

1. The chromogen formation from N-acetylneuraminic acid in the periodate-thiobarbituric acid reaction was investigated. Measurement of periodate consumption showed an uptake of approx. 3moles/mole of substrate in neutral as well as in strongly acidic solution. Therefore the chromogen beta-formylpyruvic acid is not a direct product of the periodate oxidation; it is presumed to be formed from the true oxidation product, a hexos-5-uluronic acid, by aldol splitting during the reaction in hot acidic solution with thiobarbituric acid. 2. Methyl (methyl beta-l-threo-hexos-4-enepyranosid)uronate, an analogue of the pre-chromogen, has been shown to yield with thiobarbituric acid in acidic solution a pigment exhibiting an identical absorption spectrum and showing the same behaviour on paper chromatography as the pigment obtained from N-acetylneuraminic acid in the periodate-thiobarbituric acid assay. 3. The substitution at C-2 of methoxyneuraminic acid does not inhibit the periodate-thiobarbituric acid reaction. In neutral solution methoxyneuraminic acid is oxidized by periodate to a substance that reacts readily with thiobarbituric acid in acidic solution. When periodate oxidation is attempted in acidic solution, protonation of the amino group protects this group against oxidation, rendering methoxyneuraminic acid negative in the assay systems of Warren (1959a,b) and Aminoff (1959, 1961).     

Quantitation of oligosaccharides released by the β-elimination reaction

A quantitative micromethod has been described for monitoring the rate and extent of the β-elimination reaction as applied to O-glycosyl-glycoproteins utilizing alkaline tritiated borohydride. The procedure simultaneously labels the released oligosaccharides by their reduction to the corresponding tritiated alditols. The alkaline tritiated borohydride treatment also results in the labeling of the protein moiety of the glycoprotein and this can be quantitatively separated from the carbohydrate moiety on a cation exchange resin; the carbohydrate moiety is not adsorbed, while the protein moiety is adsorbed and then eluted with HCl. The radioactivity in the aqueous eluate of the resin is therefore a direct measure of the amount of oligosaccharides released by the β-elimination reaction. The sensitivity of the method is dependent on the specific activity of the tritiated sodium borohydride used. The stoichiometry of the reaction has been established by the use of N-acetylgalactosaminyl-O-glycoproteins, demonstrating that at the completion of the β-elimination reaction: (a) none of the radioactivity attributable to the protein moiety contaminates the carbohydrate moiety, (b) all the carbohydrate components of the glycoprotein are found in the aqueous eluate from the cationic exchange resin, (c) all the radioactivity in this aqueous eluate is associated with the sugar known to be at the reducing end of the oligosaccharide chain bound to serine or threonine of the glycoprotein (in the examples discussed, N-acetylgalactosamine), and (d) there is no additional hydrolysis of the oligosaccharide chains during the processing.     

Isolation of some precursors of 2-methylpyrrole in the Elson–Morgan reaction

1-C-(1-Acetylacetonyl)-2-deoxy-2-(1-methyl-3-oxobut-1-enyl)amino -d-galactitol is obtained from the condensation of 2-amino-2-deoxy-d-galactose with pentane-2,4-dione in anhydrous solvent. On treatment with hot alkali it gives 2-methylpyrrole with 37% yield. By acid hydrolysis under mild conditions the compound loses the N substituent and from the resulting unstable derivative 2-methylpyrrole is obtained (52% yield). It is concluded that derivatives of aminohexoses substituted at C-1 with a dioxopentyl chain are the precursors of 2-methylpyrrole in the Cessi & Serafini-Cessi (1963) modification of the Elson-Morgan reaction. As demonstrated previously, products of condensation of aminohexoses with pentane-2,4-dione at the amino group are not converted directly into 2-methylpyrrole, but this step provides protection of the amino group during condensation at C-1.     

Design of new reaction conditions for characterization of a mutant thermophilic α-l-fucosidase

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.     

Reverse-docking study of the TADDOL-catalyzed asymmetric hetero-Diels–Alder reaction

The reverse-docking of a TADDOL catalyst to rigid transition-state (TS) representations of an asymmetric hetero-Diels–Alder reaction is described. The resulting docking poses represent a simplified geometric model of the TS for the catalyzed reaction. The conformational space of the catalyst in proximity to the catalyst-free TS models is sampled stochastically and the energetically favored poses are subjected to a clustering procedure to highlight structural attributes compatible with organocatalysis. Each pose is scored and ranked based on its molecular-mechanics docking energy. The reverse-docking procedure reveals a clear energetic trend in favor of the experimentally preferred product enantiomers. Analysis of the best poses suggests a geometric model that is consistent with principles of molecular recognition, catalysis, and experimental data.      

Traveling wave solutions of a nonlinear reaction–advection equation

 We establish the existence of traveling wave solutions for a nonlinear partial differential equation that models a logistically growing population whose movement is governed by an advective process. Conditions are presented for which traveling wave solutions exist and for which they are stable to small semi-finite domain perturbations. The wave is of mathematical interest because its behavior is determined by a singular differential equation and those with small speed of propagation steepen into a shock-like solutions. Finally, we indicate that the smoothing presence of diffusion allows wave persistence when an advective slow moving wave may collapse. Received: 24 November 1997 / Revised version: 13 July 1998     

Patterns of patchy spread in multi-species reaction–diffusion models

Spread of populations in space often takes place via formation, interaction and propagation of separated patches of high species density, without formation of continuous fronts. This type of spread is called a ‘patchy spread’. In earlier models, this phenomenon was considered to be a result of a pronounced environmental or/and demographic stochasticity. Recently, it was found that a patchy spread can arise in a fully deterministic predator–prey system and in models of infectious diseases; in each case the process takes place in a homogeneous environment. It is well recognized that the observed patterns of patchy spread in nature are a result of interplay between stochastic and deterministic factors. However, the models considering deterministic mechanism of patchy spread are developed and studied much less compared to those based on stochastic mechanisms. A further progress in the understanding of the role of deterministic factors in the patchy spread would be extremely helpful. Here we apply multi-species reaction–diffusion models of two spatial dimensions in a homogeneous environment. We demonstrate that patterns of patchy spread are rather common for the considered approach, in particular, they arise both in mutualism and competition models influenced by predation. We show that this phenomenon can occur in a system without a strong Allee effect, contrary to what was assumed to be crucial in earlier models. We show, as well, a pattern of patchy spread having significantly different speeds in different spatial directions. We analyze basic features of spatiotemporal dynamics of patchy spread common for the reaction–diffusion approach. We discuss in which ecosystems we would observe patterns of deterministic patchy spread due to the considered mechanism.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.     

The arsenolysis reaction in the biotechnological method of synthesis of modified purine β-D-arabinonucleosides

We found a unique property of E. coli purine nucleoside phosphorylases to selectively perform the arsenolysis reaction of ribonucleosides in their active site without affecting β-D-arabinonucleosides. In the synthesis of modified β-D-arabinonucleosides from the corresponding ribonucleosides, the catalytical amount of sodium arsenate in the transglycosylation reaction provided a 95 to 98% conversion rate. Such an approach was shown to simplify the composition of the reaction mixtures and facilitate the isolation of the target nucleosides, particularly, vidarabine, fludarabine, and nelarabine.     

The adenosine diphosphate–adenosine triphosphate-exchange reaction of cerebral microsomes and its relation to the sodium ion-stimulated adenosine-triphosphatase reaction

Microsomes from guinea-pig cerebral cortex contain a system capable of exchanging ADP with ATP at rates of about 20mumoles/mg. of protein/hr. The ADP-ATP-exchange reaction requires Mg(2+) for activity. The reaction is not stimulated by Na(+) or K(+) and is not inhibited by ouabain, in contrast with the Na(+)-plus-K(+)-stimulated adenosine triphosphatase. The pH optimum also differs from that of the adenosine triphosphatase. The ADP-ATP-exchange reaction is stimulated two- to three-fold by non-ionic, anionic and cationic detergents, even when these agents are inhibiting the adenosine-triphosphatase reaction. This reaction may represent a component of the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction but is more likely to be due to other enzyme systems present in microsomal subfractions.