Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

A general assay for antibody catalysis using acridone as a fluorescent tag.

A simple and highly sensitive catalysis assay is demonstrated based on analyzing reactions with acridonetagged compounds by thin-layer chromatography. As little as 1 pmol of product is readily visualized by its blue fluorescence under UV illumination and identified by its retention factor (Rf). Each assay requires only 10 microliters of solution. The method is reliable, inexpensive, versatile, and immediately applicable in repetitive format for screening catalytic antibody libraries. Three examples are presented: (i) the epoxidation of acridone labeled (S)-citronellol. The pair of stereoisomeric epoxides formed is resolved on the plate, which provides a direct selection method for enantioselective epoxidation catalysts. (ii) Oxidation of acridone-labeled 1-hexanol to 1-hexanal. The activity of horse liver alcohol dehydrogenase is detected. (iii) Indirect product labeling of released aldehyde groups by hydrazone formation with an acridone-labeled hydrazide. Activity of catalytic antibodies for hydrolysis of enol ethers is detected.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Rapid appearance and asymmetric distribution of glucose transporter SGTP4 at the apical surface of intramammalian-stage Schistosoma mansoni.

Adult Schistosoma mansoni blood flukes reside in the mesenteric veins of their vertebrate hosts, where they absorb immense quantities of glucose through their tegument by facilitated diffusion. Previously, we obtained S. mansoni cDNAs encoding facilitated-diffusion schistosome glucose transporter proteins 1 and 4 (SGTP1 and SGTP4) and localized SGTP1 to the basal membranes of the tegument and the underlying muscle. In this study, we characterize the expression and localization of SGTP4 during the schistosome life cycle. Antibodies specific to SGTP4 appear to stain only the double-bilayer, apical membranes of the adult parasite tegument, revealing an asymmetric distribution relative to the basal transporter SGTP1. On living worms, SGTP4 is available to surface biotinylation, suggesting that it is exposed at the hose-parasite interface. SGTP4 is detected shortly after the transformation of free-living, infectious cercariae into schistosomula and coincides with the appearance of the double membrane. Within 15 min after transformation, anti-SGTP4 staining produces a bright, patchy distribution at the surface of schistosomula, which becomes contiguous over the entire surface of the schistosomula by 24 hr after transformation. SGTP4 is not detected in earlier developmental stages (eggs, sporocysts, and cercariae) that do not possess the specialized double membrane. Thus, SGTP4 appears to be expressed only in the mammalian stages of the parasite's life cycle and specifically localized within the host-interactive, apical membranes of the tegument.

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis.

Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base, aspartate would activate a water molecule to facilitate nucleophilic attack. The amino acids involved in transition-state stabilization for cysteinylphosphate hydrolysis were confirmed by the x-ray structure of the Yersinia PTPase complexed with vanadate, a transition-state mimic that binds covalently to the active-site cysteine. Consistent with the NMR, x-ray, biochemical, and kinetic data, a unifying mechanism for catalysis is proposed.

Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.

Use of binding energy by an RNA enzyme for catalysis by positioning and substrate destabilization.

A fundamental catalytic principle for protein enzymes in the use of binding interactions away from the site of chemical transformation for catalysis. We have compared the binding and reactivity of a series of oligonucleotide substrates and products of the Tetrahymena ribozyme, which catalyzes a site-specific phosphodiester cleavage reaction: CCCUCUpA+G<-->CCCUCU-OH+GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate destabilization. The stronger binding of the all-ribose oligonucleotide product compared to an analog with a terminal 3' deoxyribose residue gives an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting the presence of a structurally well defined active site capable of precise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleotide product, despite the presence of the reactive phosphoryl group in the substrate. This destabilization is accounted for by a model in which tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucleotide substrate next to an electropositive Mg ion. As the phosphodiester bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2+ becomes strong. These strategies of "substrate destabilization" and "transition state stabilization" provide estimated rate enhancements of approximately 280- and approximately 60-fold, respectively. Analogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing reactions of phosphate esters.

Asymmetric aldol reaction with BINAM-sulfonyl polymeric organocatalyst

The BINAM-sulfonyl polymeric organocatalysts was prepared by the AIBN-promoted copolymerization of BINAM-derived sulfonamide, styrene and divinylbenzebe. The polymer catalyzed the asymmetric aldol reaction of aliphatic ketones with aromatic aldehydes to give the aldol products in up to 83% yield and with up to 95% ee. The catalysts could be recovered upt to 6 times with only a slight decrease on its activity.

Chiral β-amino alcohols as ligands for the ruthenium-catalyzed asymmetric transfer hydrogenation of N-phosphinyl ketimines

Some chiral β-amino alcohols have been evaluated as potential ligands for the ruthenium-catalyzed asymmetric transfer hydrogenation (ATH) of N-phosphinyl ketimines in isopropyl alcohol. The ruthenium complex prepared from [RuCl2(p-cymene)]2 and (1S,2R)-1-amino-2-indanol has shown to be an efficient catalyst for the ATH of several N-(diphenylphosphinyl)imines, affording the reduction products in very good isolated yields and enantiomeric excesses up to 82%. The inherent rigidity of the indane ring system present in the ligand seems to be very important to achieve good enantioselectivities.

Enantioselective alkylation of β-keto esters promoted by dimeric Cinchona-derived ammonium salts as recoverable organocatalysts

Dimeric anthracenyldimethyl-derived Cinchona ammonium salts are used as chiral organocatalysts in 5 mol% for the phase-transfer enantioselective alkylation reaction of 2-alkoxycarbonyl-1-indanones with activated bromides. The corresponding adducts bearing a new all-carbon quaternary center are obtained usually in high yield and with moderate and opposite enantioselectivity (up to 55%) when using ammonium salts derived from quinidine and its pseudoenantiomer quinine as organocatalysts. These catalysts can be almost quantitatively recovered by precipitation in ether and reused.

A Highly Efficient Solvent-Free Asymmetric Direct Aldol Reaction Organocatalyzed by Recoverable (s)-Binam-L-Prolinamides. ESI-MS Evidence of the Enamine-Iminium Formation

Recoverable (Sa)-binam-l-prolinamide in combination with benzoic acid is used as catalysts in the direct aldol reaction between cycloalkyl, alkyl, and α-functionalized ketones and aldehydes under solvent-free reaction conditions. Three different methods are assayed: simple conventional magnetic stirring, magnetic stirring after previous dissolution in THF and evaporation, and ball mill technique. These procedures allow one to reduce not only the amount of required ketone to 2 equiv but also the reaction time to give the aldol products with regio-, diastereo-, and enantioselectivities comparable to those in organic or aqueous solvents. Generally anti-isomers are mainly obtained with enantioselectivities up to 97%. The reaction can be carried out under these conditions also using aldehydes as nucleophiles, yielding after in situ reduction of the aldol products the corresponding chiral 1,3-diols with moderate to high enantioselectivities mainly as anti-isomers. The aldol reaction has been studied by the use of positive ESI-MS technique, providing the evidence of the formation of the corresponding enamine−iminium intermediates.

Economic cycles, asymmetric crises and tourism competitiveness: emerging versus mature destinations

This paper considers the influence of business cycles and economic crises on tourism destinations competitiveness. This competitiveness is measured by its share in world tourism. Analysing a period of forty years, the differential permanent or temporary effects that economic crises has on competitiveness of mature and emerging destinations are observed. Furthermore, it identifies the economic transmission mechanisms operating within this context, analysing them using the framework of the most relevant explanatory models of tourism destination competitiveness. The preliminary results obtained suggest that the effects of these shocks on competitiveness are not neutral. In mature destinations the negative effects are more persistent in highly intensive crises. In emerging destinations with a growing natural trend on tourism demand, the effects of the economic crises are softer and limited, reinforcing the process of convergence between destinations. This effect works through two basic transmission mechanisms: the reduction of internal and external tourism demand and the decrease on investment.

Carbon–carbon asymmetric aqueous capacitor by pseudocapacitive positive and stable negative electrodes

An asymmetric aqueous capacitor was constructed by employing zeolite-templated carbon (ZTC) as a pseudocapacitive positive electrode and KOH-activated carbon as a stable negative electrode. The asymmetric capacitor can be operated with the working voltage of 1.4 V, and exhibits an energy density that is comparable to those of conventional capacitors utilizing organic electrolytes, thanks to the large pseudocapacitance of ZTC. Despite relatively thick electrode (0.2 mm) configuration, the asymmetric capacitor could be well operated under a current density of 500 mA g −1.

Asymmetric hybrid capacitors based on activated carbon and activated carbon fibre–PANI electrodes

Composites consisting of polyaniline (PANI) coatings inside the microporosity of an activated carbon fibre (ACF) were prepared by electrochemical and chemical methods. Electrochemical characterization of both composites points out that the electrodes with polyaniline show a higher capacitance than the pristine porous carbon electrode. These materials have been used to develop an asymmetric capacitor based on activated carbon (AC) as negative electrode and an ACF–PANI composite as positive electrode in H2SO4 solution as electrolyte. The presence of a thin layer of polyaniline inside the porosity of the activated carbon fibres avoids the oxidation of the carbon material and the oxygen evolution reaction is produced at more positive potentials. This capacitor was tested in a maximum cell voltage of 1.6 V and exhibited high energy densities, calculated for the unpackaged active materials, with values of 20 W h kg−1 and power densities of 2.1 kW kg−1 with excellent cycle lifetime (90% during the first 1000 cycles) and high coulombic efficiency.