3,960 research outputs found
Role of the Zn\u3csub\u3e1\u3c/sub\u3e and Zn\u3csub\u3e2\u3c/sub\u3e Sites in Metallo-β-lactamase L1
In an effort to probe the role of the Zn(II) sites in metallo-β-lactamase L1, mononuclear metal ion containing and heterobimetallic analogues of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)- and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn2 site in L1. Heterobimetallic analogues (ZnCo and ZnFe) analogues of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogues were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is 5-coordinate in the resting state, proceeds through a 4-coordinate species during the reaction, and is 5-coordinate in the enzyme−product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the nitrocefin-derived reaction intermediate
Conformational Changes in the Metallo-β-lactamase ImiS During the Catalytic Reaction: An EPR Spectrokinetic Study of Co(II)-Spin Label Interactions
Metallo-β-lactamases are responsible for conferring antibiotic resistance on certain pathogenic bacteria. In consequence, the search for inhibitors that may be useful in combating antibiotic resistance has fueled much study of the active sites of these enzymes. There exists circumstantial evidence that the binding of substrates and inhibitors to metallo-β-lactamases may involve binding to the organic part of the molecule, in addition to or prior to binding to one or more active site metal ions. It has also been postulated that a conformational change may accompany this putative binding. In the present study, electron paramagnetic resonance spectrokinetic study of a spin-labeled variant of the class B2 metallo-β-lactamase ImiS identified movement of a component residue on a conserved α-helix in a catalytically competent time upon formation of a transient reaction intermediate with the substrate imipenem. In a significant subpopulation of ImiS, this conformational change was not associated with substrate binding to the active site metal ion but, rather, represents a distinct step in the reaction with ImiS. This observation has implications regarding the determinants of substrate specificity in metallo-β-lactamases and the design of potentially clinically useful inhibitors
\u3cem\u3eArabidopsis thaliana\u3c/em\u3e GLX2-1 Contains a Dinuclear Metal Binding Site, but Is Not a Glyoxalase 2
In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV–visible, EPR, and 1H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV–visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in 1H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg246 binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-β-lactamase superfamily
Spectroscopic Studies on \u3cem\u3eArabidopsis\u3c/em\u3e ETHE1, a Glyoxalase II-like Protein
ETHE1 (ethylmalonic encephalopathy protein 1) is a β-lactamase fold-containing protein that is essential for the survival of a range of organisms. In spite of the apparent importance of this enzyme, very little is known about its function or biochemical properties. In this study Arabidopsis ETHE1 was over-expressed and purified and shown to bind tightly to 1.2 ± 0.2 equivalents of iron. 1H NMR and EPR studies demonstrate that the predominant oxidation state of Fe in ETHE1 is Fe(II), and NMR studies confirm that two histidines are bound to Fe(II). EPR studies show that there is no antiferromagnetically coupled Fe(III)Fe(II) center in ETHE1. Gel filtration studies reveal that ETHE1 is a dimer in solution, which is consistent with previous crystallographic studies. Although very similar in terms of amino acid sequence to glyoxalase II, ETHE1 exhibits no thioester hydrolase activity, and activity screening assays reveal that ETHE1 exhibits low level esterase activity. Taken together, ETHE1 is a novel, mononuclear Fe(II)-containing member of the β-lactamase fold superfamily
Motion of the Zinc Ions in Catalysis by a Dizinc Metallo-β-Lactamase
We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc metallo-β-lactamase (MβL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MβL move away from each other, by ∼0.3 Å after 10 ms of reaction with nitrocefin, from 3.4 to 3.7 Å. Together with our previous characterization of the resting enzyme and its nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 Å, these data indicate a scissoring motion of the active site that accompanies the ring-opening step. The average Zn(II) coordination number of 4.5 in the resting enzyme appears to be maintained throughout the reaction with nitrocefin. This is the first direct structural information available on early stage dizinc metallo-β-lactamase catalysis
Saffman-Taylor fingers with kinetic undercooling
The mathematical model of a steadily propagating Saffman-Taylor finger in a
Hele-Shaw channel has applications to two-dimensional interacting streamer
discharges which are aligned in a periodic array. In the streamer context, the
relevant regularisation on the interface is not provided by surface tension,
but instead has been postulated to involve a mechanism equivalent to kinetic
undercooling, which acts to penalise high velocities and prevent blow-up of the
unregularised solution. Previous asymptotic results for the Hele-Shaw finger
problem with kinetic undercooling suggest that for a given value of the kinetic
undercooling parameter, there is a discrete set of possible finger shapes, each
analytic at the nose and occupying a different fraction of the channel width.
In the limit in which the kinetic undercooling parameter vanishes, the fraction
for each family approaches 1/2, suggesting that this 'selection' of 1/2 by
kinetic undercooling is qualitatively similar to the well-known analogue with
surface tension. We treat the numerical problem of computing these
Saffman-Taylor fingers with kinetic undercooling, which turns out to be more
subtle than the analogue with surface tension, since kinetic undercooling
permits finger shapes which are corner-free but not analytic. We provide
numerical evidence for the selection mechanism by setting up a problem with
both kinetic undercooling and surface tension, and numerically taking the limit
that the surface tension vanishes.Comment: 10 pages, 6 figures, accepted for publication by Physical Review
On-demand single-photon state generation via nonlinear absorption
We propose a method for producing on-demand single-photon states based on
collision-induced exchanges of photons and unbalanced linear absorption between
two single-mode light fields. These two effects result in an effective
nonlinear absorption of photons in one of the modes, which can lead to single
photon states. A quantum nonlinear attenuator based on such a mechanism can
absorb photons in a normal input light pulse and terminate the absorption at a
single-photon state. Because the output light pulses containing single photons
preserve the properties of the input pulses, we expect this method to be a
means for building a highly controllable single photon source.Comment: 5 pages, 2 figures, to appear in PRA. To be published in PR
Proposal for an IMLS Collection Registry and Metadata Repository
The University of Illinois at Urbana-Champaign proposes to design, implement, and research a collection-level registry and item-level metadata repository service that will aggregate information about digital collections and items of digital content created using funds from Institute of Museum and Library Services (IMLS) National Leadership Grants. This work will be a collaboration by the University Library and the Graduate School of Library and Information Science. All extant digital collections initiated or augmented under IMLS aegis from 1998 through September 30, 2005 will be included in the proposed collection registry. Item-level metadata will be harvested from collections making such content available using the Open Archives Initiative Protocol for Metadata Harvesting (OAI PMH). As part of this work, project personnel, in cooperation with IMLS staff and grantees, will define and document appropriate metadata schemas, help create and maintain collection-level metadata records, assist in implementing OAI compliant metadata provider services for dissemination of item-level metadata records, and research potential benefits and issues associated with these activities. The immediate outcomes of this work will be the practical demonstration of technologies that have the potential to enhance the visibility of IMLS funded online exhibits and digital library collections and improve discoverability of items contained in these resources. Experience gained and research conducted during this project will make clearer both the costs and the potential benefits associated with such services. Metadata provider and harvesting service implementations will be appropriately instrumented (e.g., customized anonymous transaction logs, online questionnaires for targeted user groups, performance monitors). At the conclusion of this project we will submit a final report that discusses tasks performed and lessons learned, presents business plans for sustaining registry and repository services, enumerates and summarizes potential benefits of these services, and makes recommendations regarding future implementations of these and related intermediary and end user interoperability services by IMLS projects.unpublishednot peer reviewe
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