611 research outputs found

    Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

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    To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in kcat. The kcat value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while kcat decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The Km values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The kcat/Km values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from ∼50- to 580-fold reduction. The pH dependence of log Km, log kcat, and log(kcat/Km) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pKa values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at λmax(640) vs pH for both WT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site

    All at sea: When duty meets austerity in scheduling monuments in english waters

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    The current policy and legal framework surrounding the scheduling of submerged monuments in English waters, its application and the implications for the conservation of underwater cultural heritage arising is reviewed. Historic England (HE's) new policy being seemingly based primarily upon the perceived implications for its resources. The resulting dichotomy of policy between DCMS and its statutory advisers is at best confusing to both the marine archeological community and the public, at worst it may be ultra vires. At times of unprecedented public funding austerity statutory agencies such as HE need to build support amongst their public constituencies. Formulating such an approach is potentially alienating and does not seen well designed to achieve this. The 1979 Act also appears to offer a versatile instrument for managing underwater sites. Indeed the Act may fit HE's public agenda better than the 1973 Act, delivering the public access that HE desires, without he resource implications generated pursuant to the granting of licenses for visiting under the 1973 Act

    Energy Audits of Local Government Buildings (Louisiana DNR)

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    This paper describes innovations instituted in an ongoing energy auditing program for local government buildings in Louisiana. In order to provide as many audits as possible with the funds available, a two-pronged effort was initiated to reduce the cost of conducting audits and generating audit reports while maintaining high quality results: (1) senior mechanical engineering students were employed and trained as auditors and (2) a microcomputer-based software system was developed to automate much of the analysis, economic decision making, and composition required for the audit reports. In addition to providing a valuable educational tool for the students and a streamlined cost effective means of generating audit reports for local governments, this project can potentially improve the energy efficiency by as much as 1,768 MMBTU/yr per building. These numbers are extrapolated from the results of this program performed in 1986 before the development and use of the microcomputer-based software system

    Evaluation of the protection against norovirus afforded by E. coli monitoring of shellfish production areas under EU regulations

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    EC Regulation 854/2004 requires the classification of bivalve mollusc harvesting areas according to the faecal pollution status of sites. It has been reported that determination of Escherichia coli in bivalve shellfish is a poor predictor of norovirus (NoV) contamination in individual samples. We explore the correlation of shellfish E. coli data with norovirus presence using data from studies across 88 UK sites (1,184 paired samples). We investigate whether current E. coli legislative standards could be refined to reduce NoV infection risk. A significant relationship between E. coli and NoV was found in the winter months (October to February) using data from sites with at least 10 data pairs (51 sites). We found that the ratio of arithmetic means (log10 E. coli to log10 NoV) at these sites ranged from 0.6 to 1.4. The lower ratios (towards 0.6) might typically indicate situations where the contribution from UV disinfected sewage discharges was more significant. Conversely, higher ratios (towards 1.4) might indicate a prevalence of animal sources of pollution; however, this relationship did not always hold true and so further work is required to fully elucidate the factors of relevance. Reducing the current class B maximum (allowed in 10% of samples) from 46,000 E. coli per 100 g (corresponding NoV value of 75750±103) to 18,000 E. coli per 100 g (corresponding NoV value of 29365±69) reduces maximum levels of NoV by a factor of 2.6 to 1; reducing the upper class B limit to 100% compliance with 4,600 E. coli per 100 g (corresponding NoV value of 7403±39) reduces maximum levels of NoV by a factor of 10.2 to 1. We found using the UK filtered winter dataset that a maximum of 200 NoV corresponded to a maximum of 128±7 E. coli per 100 g. A maximum of 1,000 NoV corresponded to a maximum of 631±14 E. coli per 100 g

    Correlated sampling in quantum Monte Carlo: a route to forces

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    In order to find the equilibrium geometries of molecules and solids and to perform ab initio molecular dynamics, it is necessary to calculate the forces on the nuclei. We present a correlated sampling method to efficiently calculate numerical forces and potential energy surfaces in diffusion Monte Carlo. It employs a novel coordinate transformation, earlier used in variational Monte Carlo, to greatly reduce the statistical error. Results are presented for first-row diatomic molecules.Comment: 5 pages, 2 postscript figure
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