Applications of Hydrolytic and Decarboxylating Enzymes in Biotransformations

Peptides, and oligosaccharides and glycosides, can be synthesised by making use of the 'reverse hydrolytic activity' of proteases and glycosidases respectively. In applying these enzymes to the practical synthesis of these classes of compound, several factors need to be considered, namely the need to shift the rate-determining step through the use of activated substrates, the need to minimise competing hydrolysis of these and the need to minimise hydrolysis of the products. In spite of these problems, the enzymatic methods have many attractive features, not least amongst which is the absolute control of stereochemistry in acyl transfer and glycosyl transfer respectively.     

Enzymic mechanisms involving concomitant transfer and hydrolysis reactions

The kinetic parameters of ten different enzymic mechanisms in which bimolecular transfer reactions occur concomitantly with the hydrolysis of the donor molecule have been studied. The usefulness of these parameters for making a choice of mechanism is discussed. The analysis has been extended to the use of alternative substrates in bimolecular transfer reactions that proceed without the hydrolysis of the donor molecule.     

Human renal gamma-glutamyltransferase hydrolysis and transfer reactions with glutathione as substrate

The hydrolysis and transfer reactions of purified human renal gamma-glutamyltransferase were studied in vitro with glutathione as substrate at pH and substrate concentration reflecting the physiological conditions. The pH optimum ranged from 7.48 to 8.44 for hydrolysis and 7.90 to 8.92 for transfer with glutamine as acceptor. The Michaelis constants for glutathione were 13 microM in hydrolysis and 58 microM in transfer reactions respectively. Inhibition of transfer occurred for glutathione concentrations above 0.4 mM. Various ions, urea, creatinine, uric acid and L-amino acids were shown to have no appreciable effect on both reactions except L-glutamine which acts as an activator on the hydrolysis activity. Taken together, our results, if they are transposable in vivo would be relevant of an enzyme acting like an hydrolase rather than like a transferase.     

Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods.

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.     

Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pre-transfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre- and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.     

Primary 13C and beta-secondary 2H KIEs for trans-sialidase. A snapshot of nucleophilic participation during catalysis

Trypanosoma cruzi trans-sialidase catalyzes a novel reaction that involves the transfer of sialic acid between host and parasite glycoconjugates. In this paper, we report kinetic isotope effect studies on recombinant trans-sialidase. beta-Dideuterium and primary 13C isotope effects were measured for a good substrate, sialyl-lactose, and a slow substrate, sialyl-galactose, in both acid-catalyzed solvolysis and enzymatic transfer reactions. The beta-dideuterium isotope effect for sialyl-lactose in the acid hydrolysis reaction was 1.113 +/- 0.012. The primary 13C isotope effects for hydrolysis of sialyl-lactose and sialyl-galactose were 1. 016 +/- 0.011 and 1.015 +/- 0.008, respectively. In the enzymatic transfer reactions, the beta-dideuterium and primary 13C effects for sialyl-galactose were 1.060 +/- 0.008 and 1.032 +/- 0.008, respectively. The isotope effects for hydrolysis describe a dissociative SN1-like mechanism, and these data are contrasted by the data for the enzyme-catalyzed reaction. The enzymatic deuterium isotope effects are lower by a factor of 2, but the primary carbon isotope effects are higher by a factor of 2. This pattern describes a mechanism involving nucleophilic participation in the rate-determining transition state.     

Isolation and properties of cellobiase from Penicillium verruculosum]

Cellobiase (beta-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i.e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose-delta-lactone competitively inhibited cellobiase (Ki = 0.19 mM and 17 microM, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.     

The reaction of 1,2:5,6-di-O-isopropylidene-α-d-ribo-hexofuranos-3-ulose with diazomethane

Dextransucrase was shown to catalyze the hydrolysis of sucrose. The hydrolytic activity was found to be directly correlatable with dextransucrase activity on poly-(acrylamide) disc-gel electrophoresis. In studies on the hydrolysis of sucrose and formation of dextran as a function of time and substrate concentration, the two activities were found to be competitive with each other. Competition was also observed between hydrolysis and the transfer of d-glucosyl groups to added acceptors. The results suggest that the three activities, namely, polymerization, d-glucosyl transfer, and hydrolysis, compete for a form of the enzyme that is common to all three reactions. It is proposed that this form may be a d-glucosylated derivative of the enzyme.     

Targeting neuronal nitric oxide synthase with gene transfer to modulate cardiac autonomic function

Microdomains of neuronal nitric oxide synthase (nNOS) are spatially localised within both autonomic neurons innervating the heart and post-junctional myocytes. This review examines the use of gene transfer to investigate the role of nNOS in cardiac autonomic control. Furthermore, it explores techniques that may be used to improve upon gene delivery to the cardiac autonomic nervous system, potentially allowing more specific delivery of genes to the target neurons/myocytes. This may involve modification of the tropism of the adenoviral vector, or the use of alternative viral and non-viral gene delivery mechanisms to minimise potential immune responses in the host.

Here we show that adenoviral vectors provide an efficient method of gene delivery to cardiac–neural tissue. Functionally, adenovirus-nNOS can increase cardiac vagal responsiveness by facilitating cholinergic neurotransmission and decrease β-adrenergic excitability. Whether gene transfer remains the preferred strategy for targeting cardiac autonomic impairment will depend on site-specific promoters eliciting sustained gene expression that results in restoration of physiological function.     

A neurochemical approach for studying response to acetylcholine in Alzheimer's disease

The proposal of cholinomimetic treatment as a rational basis for the therapy of Alzheimer's disease has been prematurely dismissed by some workers on the hypothesis of impaired coupling/signal transduction of postsynaptic cholinergic receptors. Disparity of reports studying such impairment may be due to inappropriate extrapolation of experimental systems to the physiological stituation, as well as inadequate consideration of disease epiphenoma. In the present study we have used samples with short duration of terminal coma, collected using techniques to minimise postmortem autolysis, and samples obtained during neurosurgery to examine carbachol stimulated hydrolysis of [³H]phosphatidylinositol (PI) as a marker for receptor/signal transduction integrity. The influence of postmortem delay was also studied using another series of samples and a rat model. While a significant correlation of postmortem delay and carbachol stimulated [³H]PI hydrolysis was found, comparison of pooled neurosurgical and postmortem controls with AD samples revealed no significant reduction. Thus this study concurs with a similar one previously reported here, using [³H]phosphatidylinositol 4,5-bisphosphate (1). They provide evidence for competent receptor-signal transduction events in AD, supporting the use of cholinomimetic therapy for disease treatment.     

Characterization of reconstituted ATPase complex proteoliposomes prepared from the thermophilic cyanobacterium Synechococcus 6716

The preparation and some properties are described of proteoliposomes consisting of the ATPase complex and lipids from the thermophilic cyanobacterium Synechococcus 6716. In the proteoliposomes (about 200 nm in diameter) only a low amount of protein can be incorporated (protein/lipid ratio of 0.01 w/w) and they show very few protein particles on freeze-fracture replicas. The octyl glucoside and cholate dialysis method of reconstitution yielded stable proteoliposomes with a relatively low proton permeability. ATP hydrolysis and 32Pi/ATP exchange activities were about 400 and 120 nmol X min-1 X mg protein-1, respectively; the former was strongly stimulated by an uncoupler. ATP hydrolysis induces membrane energization as monitored by membrane-potential- and surface-potential-indicating probes and by different pH indicators trapped inside the vesicles. The probes used were a membrane-bound fluorescent aminoacridine, which monitors surface charge-density changes, the native carotenoids and added oxonol VI for monitoring electrical potential in the membrane and the pH indicators neutral red and cresol red. The different rise kinetics of these probes indicate that proton accumulation upon ATP hydrolysis involves at least two steps: a membrane-localized potential charge and proton transfer followed by a much slower acidification of the bulk intravesicular space. Internal neutral red and cresol red seem to discriminate between proton translocation to the internal interface and bulk space, respectively.     

Kinetics of sequential reaction of hydrolysis and sugar degradation of rice husk in ethanol production: effect of catalyst concentration

This study focuses on kinetics of rice husk hydrolysis using sulfuric acid catalyst to produce sugars. The experiments were conducted at various catalyst concentrations. It turned out that during hydrolysis, degradation of sugars was encountered. The kinetics was expressed with both homogeneous and heterogeneous models. At catalyst concentration of higher than 0.44 N, heterogeneous model works better than homogeneous model, while at the lower, both models work well. In the heterogeneous model, it is observed that the mass transfer of sulfuric acid in the particles and the hydrolysis reaction control the rate of hydrolysis. The mass transfer can be described by Fick's law with the effective diffusivity of 1.4×10(-11) cm2/s, while the hydrolysis and sugar degradation rate constants follow Arrhenius equations. In addition, it was experimentally observed that the sugars produced can be converted to ethanol by fermentation using yeast.     

Influence of temperature on protein hydrolysis in a cyclic batch enzyme membrane reactor

A cyclic batch enzyme membrane reactor (CBEMR) incorporating a 8000-Da polyethersulphone membrane was intended for enhancing the enzyme (Protex 6L from Bacillus licheniformis, EC. 3.4.21.62) use in the production of a whey protein hydrolysate. A mechanistic mathematical model comprising zero-order kinetics for the hydrolysis and second-order deactivation for the enzyme was proposed and validated through experiments. The influence of reaction temperature was studied and process optimisation (given the production requirements) was performed in terms of number of batch reactions that minimise the total amount of enzyme used. The optimal operation of the CBEMR allowed savings of up to 44 and 32% of enzyme compared to the single batch operation mode at 50 and 60 °C, respectively. No enzyme savings were detected when temperature was fixed at 70 °C. In general, the optimal operation temperature was 60 °C, yielding lower enzyme consumption for all productivities of the reactor.     

Studies on structure-based sequence alignment and phylogenies of beta-lactamases

The β-lactamases enzymes cleave the amide bond in β-lactam ring, rendering β-lactam antibiotics harmless to bacteria. In this communication we have studied structure-function relationship and phylogenies of class A, B and D beta-lactamases using structure-based sequence alignment and phylip programs respectively. The data of structure-based sequence alignment suggests that in different isolates of TEM-1, mutations did not occur at or near sequence motifs. Since deletions are reported to be lethal to structure and function of enzyme. Therefore, in these variants antibiotic hydrolysis profile and specificity will be affected. The alignment data of class A enzyme SHV-1, CTX-M-15, class D enzyme, OXA-10, and class B enzyme VIM-2 and SIM-1 show sequence motifs along with other part of polypeptide are essentially conserved. These results imply that conformations of betalactamases are close to native state and possess normal hydrolytic activities towards beta-lactam antibiotics. However, class B enzyme such as IMP-1 and NDM-1 are less conserved than other class A and D studied here because mutation and deletions occurred at critically important region such as active site. Therefore, the structure of these beta-lactamases will be altered and antibiotic hydrolysis profile will be affected. Phylogenetic studies suggest that class A and D beta-lactamases including TOHO-1 and OXA-10 respectively evolved by horizontal gene transfer (HGT) whereas other member of class A such as TEM-1 evolved by gene duplication mechanism. Taken together, these studies justify structure-function relationship of beta-lactamases and phylogenetic studies suggest these enzymes evolved by different mechanisms.     

Current strategies in the search for novel antiparasitic agents

The market for antiparasitic products comprises the largest segment for sales of livestock and companion-animal healthcare agents. Despite the availability of highly effective, broad-spectrum agents, there remains a need for safer, more convenient and more environmentally friendly products that will overcome the ever-present threat of resistance development. The very high cost of discovering and developing a new drug, especially for use in livestock, is reflected in the limited number of new classes of antiparasitic agent launched on the market. New strategies are being adopted to minimise the cost of discovering potential drug candidates by maximising the chance of identifying a useful target mechanism of action and by speeding the time to discover and optimise a lead structure. These rely heavily on new technologies in target identification, screen development and lead optimisation. Examples of these will be discussed and speculation made about the possible factors that could influence the future shape of antiparasitic control.     

Stress transfer in cellulose nanowhisker composites--influence of whisker aspect ratio and surface charge

The mechanically induced molecular deformation of cellulose nanowhiskers embedded in subpercolation concentration in an epoxy resin matrix was monitored through Raman spectroscopy. Cellulose nanowhiskers isolated by sulfuric acid hydrolysis from tunicates and by sulfuric acid hydrolysis and hydrochloric acid hydrolysis from cotton were used to study how the aspect ratio (ca. 76 for tunicate and 19 for cotton) and surface charges (38 and 85 mmol SO(4)(-)/kg for sulfuric acid hydrolysis of cotton and tunicate, respectively; no detectable surface charges for hydrochloric acid hydrolysis) originating from the isolation process influence stress transfer in such systems. Atomic force microscopy confirmed that uncharged cellulose nanowhiskers produced by hydrochloric acid hydrolysis have a much higher tendency to aggregate than the charged cotton or tunicate nanowhiskers. Each of these nanowhisker types was incorporated in a concentration of 0.7 vol % in a thermosetting epoxy resin matrix. Mechanically induced shifts of the Raman peak initially located at 1095 cm(-1) were used to express the level of deformation imparted to the nanowhiskers embedded in the resin. Much larger shifts of the diagnostic Raman band were observed for nanocomposites with tunicate nanowhiskers than for the corresponding samples comprising cotton nanowhiskers. In the case of nanocomposites comprising nanowhiskers produced by hydrochloric acid hydrolysis, no significant Raman band shift was observed. These results are indicative of different modes of stress transfer, which in turn appear to originate from the different sample morphologies.     

Clinical trial design in chronic obstructive pulmonary disease: current perspectives and considerations with regard to blinding of tiotropium

Randomised, double-blind, controlled trials are considered the gold standard for evaluating a pharmacological agent, as they minimise any potential bias. However, it is not always possible to perform double-blind trials, particularly for medications delivered via specific devices, e.g. inhalers. In such cases, open-label studies can be employed instead. Methods used to minimise any potential bias introduced by open-label study design include randomisation, crossover study design, and objective measurements of primary efficacy and safety variables. Concise reviews analysing the effect of blinding procedures of comparator drugs on outcomes in respiratory trials are limited. Here, we compare data from different chronic obstructive pulmonary disease trials with once-daily indacaterol versus a blinded or non-blinded comparator. The clinical trial programme for indacaterol, a once-daily, long-acting β₂-agonist, used tiotropium as a comparator either in an open-label or blinded fashion. Data from these studies showed that the effects of tiotropium were consistent for forced expiratory volume in 1 second, an objective measure, across blinded and non-blinded studies. The data were consistent with previous studies of double-blind tiotropium, suggesting that the open-label use of tiotropium did not introduce treatment bias. The effect of tiotropium on subjective measures (St George’s Respiratory Questionnaire; transition dyspnoea index) varied slightly across blinded and non-blinded studies, indicating that minimal bias was introduced by using open-label tiotropium. Importantly, the studies used randomised, open-label tiotropium patients to treatment allocation, a method shown to minimise bias to a greater degree than blinding. In conclusion, it is important when reporting a clinical trial to be transparent about who was blinded and how the blinding was performed; if the design is open-label, additional efforts must be made to minimise risk of bias. If these recommendations are followed, and the data are considered in the full knowledge of any potential sources of bias, results with tiotropium suggest that data from open-label studies can provide valuable and credible evidence of the effects of therapy.     

Embryo manipulation in research and animal production

Research in developmental biology has resulted in techniques to accelerate changes in gene frequency and to interfere directly in the genome. Procedures already in use or being adapted to livestock include embryo transfer, chimera production, embryo splitting, gene transfer and nuclear transplantation. Experiments with mouse embryos are revealing the principles governing embryonic development and differentiation and illustrate the need for these investigations to be extended to embryos of livestock. The optimal combination of these technologies in animal production strategies will depend upon further research and the role of animal products in society.     

Effective use of a Limited Antiviral Stockpile for Pandemic Influenza

Just allocation of resources for control of infectious diseases can be profoundly influenced by the dynamics of those diseases. In this paper we discuss the use of antiviral drugs for treatment of pandemic influenza. While the primary effect of such drugs is to alleviate and shorten the duration of symptoms for treated individuals, they can have a secondary effect of reducing transmission in the community. However, existing stockpiles may be insufficient for all clinical cases. Here we use simple mathematical models to present scenarios where the optimum policies to minimise morbidity and mortality, with a limited drug stockpile, are not always the most intuitively obvious and may conflict with theories of justice. We discuss ethical implications of these findings.     

Pyridoxal isonicotinoyl hydrazone and analogues

Summary The ultraviolet-visible absorption spectra of the orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), and three analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxalp-methoxybenzoyl hydrazone (PpMBH) and pyridoxalm-fluorobenzoyl hydrazone (PmFBH) have been measured in aqueous solution with various concentrations of added acid or alkali. Assignment of absorption bands to various molecular species in equilibrium in aqueous solution is made by reference to their acid ionisation constants. All four hydrazones were stable at physiologial pH, but hydrolysed in strongly acidic and basic solutions, resulting in the liberation of pyridoxal and the acid hydrazide. In acidic solutions this resulted in a dramatic decrease in the intensity of absorption at wavelengths of 225 nm and above 300 nm, allowing a quantitative estimate of the degree of acid-catalysed hydrolysis of the ligands. These results indicate that for oral administration the chelator should be administered with calcium carbonate or provided with an enteric coating to minimise acid-catalysed hydrolysis in the stomach. At high pH, base-catalysed hydrolysis occurred, resulting in a decrease in the absorption at a wavelength of 387 run.