430 research outputs found

    A new intrinsic thermal parameter for enzymes reveals true temperature optima

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    Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, Teq, arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source (“evolved”) temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on Teq with respect to enzyme temperature/activity effects to be more important than on thermal stability. Teq is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution

    Evidence for a third thermal parameter of enzymes

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    This thesis describes tests of a new hypothesis describing the effect of temperature on enzyme activity. Traditionally, the dependence of enzyme activity on temperature has been described by a model (the 'Classical Model') consisting of two processes: the catalytic reaction defined by ΔG‡cat, and irreversible inactivation defined by ΔG‡inact. To account for the anomalies found in the variation of enzyme activity with temperature, a new model (the 'Equilibrium Model') has been formulated to describe the effect of temperature on enzyme activity. In addition to the processes described by ΔG‡cat and ΔG‡inact, this model incorporates an inactive (but not denatured) form of the enzyme (Einact) that is in reversible equilibrium with the active form (Eact). The equilibrium between Eact and Einact is described by an equilibrium constant (Keq), whose temperature dependence is characterised in terms of the enthalpy of the equilibrium, ΔHeq, and a new thermal parameter, Teq, which is the temperature at which the concentrations of Eact and Einact are equal. This research has set out to: test the 'Equilibrium Model'; investigate the molecular basis of the temperature-dependent interconversion of the active and inactive forms of the enzyme; develop methods for the reliable determination of Teq, and outline the assay parameters required for accurate determination of Teq; examine the biotechnological implications of Teq; and finally, examine the evolutionary and ecological implications of Teq The 'Equilibrium Model' was tested by comparing 3D plots of experimental data (expressed as rate versus temperature versus time) collected for five enzymes with the 3D plots of the outputs predicted by the 'Classical' and 'Equilibrium' models. This analysis found that all five enzymes behaved as predicted by the 'Equilibrium Model', in displaying clear temperature optima at time zero, and led to the determination of plausible values for ΔG‡cat, ΔG‡inact, ΔHeq, and Teq. The value of Teq was affected when the enzyme-substrate interaction was altered (by the use of different substrates) but was, in general, unaffected by the addition of denaturing or stabilising agents to the assay. These results give some insight into the molecular basis of the equilibrium and, together with the fast timescale with which the Eact/Einact equilibrium occurs, support the hypothesis that it is unlikely that Einact is significantly unfolded and that the transition from Eact to Einact involves only a local (reversible) conformational change, possibly near or at the active site; in contrast to the slower and largely irreversible (under assay conditions) global conformational changes associated with thermal denaturation. The methodology for the determination of Teq was developed and extended to allow less laborious determination of Teq and to allow the determination of Teq using assay systems that are less than "ideal". Minimum assay parameters were determined in terms of sampling rate and temperature range. The potential implications of Teq for enzyme evolution, protein engineering and enzyme reactor performance have been introduced and discussed. The characterisation of three enzymes, once each from a psychrophilic, mesophilic and thermophilic source, has given some indication of the ecological and environmental implications of Teq, and suggests that Teq is a better indication of the source temperature of the enzyme than thermal stability and that ΔHeq reflects the thermal environment from which the enzyme was sourced. The results of this research are such that Teq can now be considered a fundamental thermal parameter of enzymes and is required alongside the Arrhenius activation energy and thermal stability to completely account for the way in which enzyme activity behaves with respect to temperature

    Matlab application for fitting progress curves to the Equilibrium Model

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    The general procedures for carrying out the necessary rate determinations required for accurate determination of the Equilibrium Model parameters, and fitting this data to the mathematical model to generate the parameters, are described in "Peterson, M.E., Daniel, R.M., Danson, M.J. & Eisenthal, R. (2007) The dependence of enzyme activity on temperature: determination and validation of parameters. Biochemical Journal, 402, 331-337". It should be borne in mind that the Equilibrium Model equation contains exponentials of exponentials – quite small deviations from ideal behaviour, or a failure to obtain true Vmax values, may lead to difficulty in obtaining reliable Equilibrium Model parameters

    Use of waveform lidar and hyperspectral sensors to assess selected spatial and structural patterns associated with recent and repeat disturbance and the abundance of sugar maple (Acer saccharum Marsh.) in a temperate mixed hardwood and conifer forest.

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    Abstract Waveform lidar imagery was acquired on September 26, 1999 over the Bartlett Experimental Forest (BEF) in New Hampshire (USA) using NASA\u27s Laser Vegetation Imaging Sensor (LVIS). This flight occurred 20 months after an ice storm damaged millions of hectares of forestland in northeastern North America. Lidar measurements of the amplitude and intensity of ground energy returns appeared to readily detect areas of moderate to severe ice storm damage associated with the worst damage. Southern through eastern aspects on side slopes were particularly susceptible to higher levels of damage, in large part overlapping tracts of forest that had suffered the highest levels of wind damage from the 1938 hurricane and containing the highest levels of sugar maple basal area and biomass. The levels of sugar maple abundance were determined through analysis of the 1997 Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) high resolution spectral imagery and inventory of USFS Northern Research Station field plots. We found a relationship between field measurements of stem volume losses and the LVIS metric of mean canopy height (r2 = 0.66; root mean square errors = 5.7 m3/ha, p \u3c 0.0001) in areas that had been subjected to moderate-to-severe ice storm damage, accurately documenting the short-term outcome of a single disturbance event

    Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome

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    Background The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. Results To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation, respiratory metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle. Conclusions The observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play key roles in host-pathogen interactions, signal transduction during conidial germination, or survival in hostile environments

    Rapid increases in soil pH solubilise organic matter, dramatically increase denitrification potential and strongly stimulate microorganisms from the Firmicutes phylum

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    Rapid and transient changes in pH frequently occur in soil, impacting dissolved organic matter (DOM) and other chemical attributes such as redox and oxygen conditions. Although we have detailed knowledge on microbial adaptation to long-term pH changes, little is known about the response of soil microbial communities to rapid pH change, nor how excess DOM might affect key aspects of microbial N processing. We used potassium hydroxide (KOH) to induce a range of soil pH changes likely to be observed after livestock urine or urea fertilizer application to soil. We also focus on nitrate reductive processes by incubating microcosms under anaerobic conditions for up to 48 h. Soil pH was elevated from 4.7 to 6.7, 8.3 or 8.8, and up to 240-fold higher DOM was mobilized by KOH compared to the controls. This increased microbial metabolism but there was no correlation between DOM concentrations and CO2 respiration nor N-metabolism rates. Microbial communities became dominated by Firmicutes bacteria within 16 h, while few changes were observed in the fungal communities. Changes in N-biogeochemistry were rapid and denitrification enzyme activity (DEA) increased up to 25-fold with the highest rates occurring in microcosms at pH 8.3 that had been incubated for 24-hour prior to measuring DEA. Nitrous oxide reductase was inactive in the pH 4.7 controls but at pH 8.3 the reduction rates exceeded 3,000 ng N2–N g−1 h−1 in the presence of native DOM. Evidence for dissimilatory nitrate reduction to ammonium and/or organic matter mineralisation was observed with ammonium increasing to concentrations up to 10 times the original native soil concentrations while significant concentrations of nitrate were utilised. Pure isolates from the microcosms were dominated by Bacillus spp. and exhibited varying nitrate reductive potential

    FORENSIC SCIENCES Development of a Human DNA Quantitation System

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    The AluQuantℱ Human DNA Quantitation System has been developed for human-specific quantitation of forensic samples. This system uses probes specific to repetitive genetic elements allowing quantitation without target amplification. Target immobilization is unnecessary with employment of solution hybridization. The AluQuantℱ Human DNA Quantitation System uses a series of enzymatic reactions to produce a luminescent signal proportional to the quantity of human DNA present. This report demonstrates a range of quantitation from 0.1-50 ng of human DNA. Signal from non-human DNAs tested was insignificant and addition of non-human DNAs into a human sample did not alter quantitation. Lastly, the system was unaffected by degradation of sample through sonication. The AluQuantℱ Human DNA Quantitation System is a simple and sensitive method for quantitating the concentration of human DNA in forensic samples
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