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Articles 481 - 510 of 2524

Full-Text Articles in Physical Sciences and Mathematics

A Modified Version Of The Lewellen And Badrinath Measure Of Tobin's Q, Darrell Lee, James Tompkins Mar 2015

A Modified Version Of The Lewellen And Badrinath Measure Of Tobin's Q, Darrell Lee, James Tompkins

James Tompkins

Lewellen and Badrinath (1997) propose a superior method of measuring Tobin's Q. Unfortunately, their method is prone to a high percentage of missing observations and results in selecting samples of larger and more mature firms with lower Q statistics. A slight modification is proposed that preserves the appeal of their method, yet almost doubles the sample size, avoids sampling problems, and is statistically indistinguishable from their Q measure. In addition, a step in the Lewellen and Badrinath Q calculation is clarified, which was inadvertently omitted in their explanation, and, if left undone, can result in downward-biased measures of Q.

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Data Management And Visualization Of X-Ray Diffraction Spectra From Thin Film Ternary Composition Spreads, I. Takeuchi, C. Long, O. Famodu, M. Murakami, Jason Hattrick-Simpers, G. Rubloff, M. Stukowski, K. Rajan Mar 2015

Data Management And Visualization Of X-Ray Diffraction Spectra From Thin Film Ternary Composition Spreads, I. Takeuchi, C. Long, O. Famodu, M. Murakami, Jason Hattrick-Simpers, G. Rubloff, M. Stukowski, K. Rajan

Jason R. Hattrick-Simpers

We discuss techniques for managing and visualizing x-ray diffraction spectrum data for thin film composition spreads which map large fractions of ternary compositional phase diagrams. An in-house x-ray microdiffractometer is used to obtain spectra from over 500 different compositions on an individual spread. The MATLAB software is used to quickly organize the data and create various plots from which one can quickly grasp different information regarding structural and phase changes across the composition spreads. Such exercises are valuable in rapidly assessing the “overall” picture of the structural evolution across phase diagrams before focusing in on specific composition regions for detailed …

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The Effect Of Copt Crystallinity And Grain Texturing On Properties Of Exchange-Coupled Fe/Copt Systems, H. Oguchi, A. Zambano, M. Yu, Jason Hattrick-Simpers, D. Banerjee, Y. Liu, Z. Wang, J. Liu, S. Lofland, D. Josell, I. Takeuchi Mar 2015

The Effect Of Copt Crystallinity And Grain Texturing On Properties Of Exchange-Coupled Fe/Copt Systems, H. Oguchi, A. Zambano, M. Yu, Jason Hattrick-Simpers, D. Banerjee, Y. Liu, Z. Wang, J. Liu, S. Lofland, D. Josell, I. Takeuchi

Jason R. Hattrick-Simpers

The effect of the crystallinity and the grain texturing of CoPt hard layers on exchange coupled Fe/CoPt soft/hard magnetic systems was studied using gradient thickness multilayer thin films. We have studied the hard layer structures by transmission electron microscopy and x-ray diffraction, and characterized the exchange coupling interaction through magnetization loops obtained by the magneto-optical Kerr effect measurement. We found that exchange coupling strongly depends on the crystalline characteristics of the CoPt hard layer. There is correlation between the mixture of different grain orientations of the CoPt hard layer and coupling efficiency. In particular, interlayer coupling is enhanced when there …

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Rapid Structural Mapping Of Ternary Metallic Alloy Systems Using The Combinatorial Approach And Cluster Analysis, C. Long, Jason Hattrick-Simpers, M. Murakami, R. Srivastava, I. Takeuchi, V. Karen, X. Li Mar 2015

Rapid Structural Mapping Of Ternary Metallic Alloy Systems Using The Combinatorial Approach And Cluster Analysis, C. Long, Jason Hattrick-Simpers, M. Murakami, R. Srivastava, I. Takeuchi, V. Karen, X. Li

Jason R. Hattrick-Simpers

We are developing a procedure for the quick identification of structural phases in thin film composition spread experiments which map large fractions of compositional phase diagrams of ternary metallic alloy systems. An in-house scanning x-ray microdiffractometer is used to obtain x-ray spectra from 273 different compositions on a single composition spread library. A cluster analysissoftware is then used to sort the spectra into groups in order to rapidly discover the distribution of phases on the ternary diagram. The most representative pattern of each group is then compared to a database of known structures to identify known phases. Using this method, …

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Interphase Exchange Coupling In Fe/Sm-Co Bilayers With Gradient Fe Thickness, Ming-Hui Yu, Jason Hattrick-Simpers, Ichiro Takeuchi, Jing Li, Z. Wang, J. Liu, S. Lofland, Somdev Tyagi, J. Freeland, D. Giubertoni, M. Bersani, M. Anderle Mar 2015

Interphase Exchange Coupling In Fe/Sm-Co Bilayers With Gradient Fe Thickness, Ming-Hui Yu, Jason Hattrick-Simpers, Ichiro Takeuchi, Jing Li, Z. Wang, J. Liu, S. Lofland, Somdev Tyagi, J. Freeland, D. Giubertoni, M. Bersani, M. Anderle

Jason R. Hattrick-Simpers

We have fabricated Fe∕Sm–Co bilayers with gradient Fe thicknesses in order to systematically study the dependence of exchange coupling on the thickness of the Fe layer. The Fe layer was deposited at two different temperatures (150 and 300°C) to study the effect of deposition temperature on the exchange coupling. Magneto-optical Kerr effect and x-ray magnetic circular dichroism (XMCD) have been employed as nondestructive rapid characterization tools to map the magnetic properties of the gradient samples. Systematic enhancement in exchange coupling between the soft layer and the hard layer is observed as the soft layer thickness is decreased. Separate exchange couplings …

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High-Throughput Screening Of Shape Memory Alloy Thin-Film Spreads Using Nanoindentation, Arpit Dwivedi, Thomas Wyrobek, Oden Warren, Jason Hattrick-Simpers, Olubenga Famodu, Ichiro Takeuchi Mar 2015

High-Throughput Screening Of Shape Memory Alloy Thin-Film Spreads Using Nanoindentation, Arpit Dwivedi, Thomas Wyrobek, Oden Warren, Jason Hattrick-Simpers, Olubenga Famodu, Ichiro Takeuchi

Jason R. Hattrick-Simpers

We have demonstrated the utility of nanoindentation as a rapid characterization tool for mapping shape memoryalloy compositions in combinatorial thin-film libraries. Nanoindentation was performed on Ni–Mn–Al ternary composition spreads. The indentation hardness and the reduced elastic modulus were mapped across a large fraction of the ternary phase diagram. The large shape memoryalloy composition region, located around the Heusler composition (Ni2MnAl), was found to display significant departure in these mechanical properties from the rest of the composition spread. In particular, the modulus and the hardness values are lower for the martensite region than those of the rest of the phase diagram.

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Note: A Simple Thermal Gradient Annealing Unit For The Treatment Of Thin Films, C. Metting, Johnathan Bunn, Ellen Underwood, Yihao Zhu, G. Koley, T. Crawford, Jason Hattrick-Simpers Mar 2015

Note: A Simple Thermal Gradient Annealing Unit For The Treatment Of Thin Films, C. Metting, Johnathan Bunn, Ellen Underwood, Yihao Zhu, G. Koley, T. Crawford, Jason Hattrick-Simpers

Jason R. Hattrick-Simpers

A gradient annealing cell has been developed for the high-throughput study of thermalannealing effects on thin-film libraries in different environments. The inexpensive gradientannealing unit permits temperature gradients as large as 28 °C/mm and can accommodate samples ranging in length from 13 mm to 51 mm. The system was validated by investigating the effects of annealing temperature on the crystallinity, resistivity, and transparency of tin-doped indium oxide deposited on a glass substrate by magnetron sputtering. The unit developed in this work will permit the rapid optimization of materials properties such as crystallinity, homogeneity, and conductivity across a variety of applications.

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Characterization Of Magnetic Degradation Mechanism In A High Neutron-Flux Environment, Adib Samin, Jie Qiu, Jason Hattrick-Simpers, Liyang Dai-Hattrick, Yuan Zheng, Lei Cao Mar 2015

Characterization Of Magnetic Degradation Mechanism In A High Neutron-Flux Environment, Adib Samin, Jie Qiu, Jason Hattrick-Simpers, Liyang Dai-Hattrick, Yuan Zheng, Lei Cao

Jason R. Hattrick-Simpers

No abstract provided.

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Optical Cell For Combinatorial In Situ Raman Spectroscopic Measurements Of Hydrogen Storage Materials At High Pressures And Temperatures, Jason Hattrick-Simpers, Wilbur Hurst, Sesha Srinivasan, James Maslar Mar 2015

Optical Cell For Combinatorial In Situ Raman Spectroscopic Measurements Of Hydrogen Storage Materials At High Pressures And Temperatures, Jason Hattrick-Simpers, Wilbur Hurst, Sesha Srinivasan, James Maslar

Jason R. Hattrick-Simpers

An optical cell is described for high-throughput backscattering Raman spectroscopic measurements of hydrogen storagematerials at pressures up to 10 MPa and temperatures up to 823 K. High throughput is obtained by employing a 60 mm diameter × 9 mm thick sapphire window, with a corresponding 50 mm diameter unobstructed optical aperture. To reproducibly seal this relatively large window to the cell body at elevated temperatures and pressures, a gold o-ring is employed. The sample holder-to-window distance is adjustable, making this cell design compatible with optical measurement systems incorporating lenses of significantly different focal lengths, e.g., microscope objectives and single element …

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An Infrared Imaging Method For High-Throughput Combinatorial Investigation Of Hydrogenation-Dehydrogenation And New Phase Formation Of Thin Films, H. Oguchi, Jason Hattrick-Simpers, I. Takeuchi, E. Heilweil, L. Bendersky Mar 2015

An Infrared Imaging Method For High-Throughput Combinatorial Investigation Of Hydrogenation-Dehydrogenation And New Phase Formation Of Thin Films, H. Oguchi, Jason Hattrick-Simpers, I. Takeuchi, E. Heilweil, L. Bendersky

Jason R. Hattrick-Simpers

We have developed an infrared imaging setup enabling in situ infrared images to be acquired, and expanded on capabilities of an infrared imaging as a high-throughput screening technique, determination of a critical thickness of a Pd capping layer which significantly blocks infrared emission from below, enhancement of sensitivity to hydrogenation and dehydrogenation by normalizing raw infrared intensity of a Mg thin film to an inert reference, rapid and systematic screening of hydrogenation and dehydrogenation properties of a Mg–Ni composition spread covered by a thickness gradient Pd capping layer, and detection of formation of a Mg2Si phase in a Mg thin …

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High-Resolution Image Reconstruction From Digital Video By Exploitation Of Nonglobal Motion, Timothy Tuinstra, Russell Hardie Mar 2015

High-Resolution Image Reconstruction From Digital Video By Exploitation Of Nonglobal Motion, Timothy Tuinstra, Russell Hardie

Russell C. Hardie

Many imaging systems utilize detector arrays that do not sample the scene according to the Nyquist criterion. As a result, the higher spatial frequencies admitted by the optics are aliased. This creates undesirable artifacts in the imagery. Furthermore, the blurring effects of the optics and the finite detector size also degrade the image quality. Several approaches for increasing the sampling rate of imaging systems have been suggested in the literature. We propose an algorithm for resolution enhancement that exploits object motion in digital video sequences. Unlike previously defined techniques, we use an automated segmentation method to isolate rigid moving objects. …

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The High-Resolution Structures Of The Neutral And The Low Ph Crystals Of Aminopeptidase From Aeromonas Proteolytica, William Desmarais, David Bienvenue, Krzysztof Bzymek, Gregory Petsko, Dagmar Ringe, Richard Holz Mar 2015

The High-Resolution Structures Of The Neutral And The Low Ph Crystals Of Aminopeptidase From Aeromonas Proteolytica, William Desmarais, David Bienvenue, Krzysztof Bzymek, Gregory Petsko, Dagmar Ringe, Richard Holz

Richard C. Holz

The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-Å resolution at neutral pH and at 1.24-Å resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases.

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Spectroscopically Distinct Cobalt(Ii) Sites In Heterodimetallic Forms Of The Aminopeptidase From Aeromonas Proteolytica:  Characterization Of Substrate Binding, Brian Bennett, Richard Holz Mar 2015

Spectroscopically Distinct Cobalt(Ii) Sites In Heterodimetallic Forms Of The Aminopeptidase From Aeromonas Proteolytica:  Characterization Of Substrate Binding, Brian Bennett, Richard Holz

Richard C. Holz

The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy. EPR spectra of the high-spin S = 3/2 Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicated that each metal binding site provides a spectroscopically distinct signature. For [CoZn(AAP)], subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent. The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with geff values of 2.20, 3.92, and …

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Acrylamide Production Using Encapsulated Nitrile Hydratase From Pseudonocardia Thermophila In A Sol–Gel Matrix, Salette Martinez, Misty Kuhn, James Russell, Richard Holz, Timothy Elgren Mar 2015

Acrylamide Production Using Encapsulated Nitrile Hydratase From Pseudonocardia Thermophila In A Sol–Gel Matrix, Salette Martinez, Misty Kuhn, James Russell, Richard Holz, Timothy Elgren

Richard C. Holz

The cobalt-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase) was successfully encapsulated in tetramethyl orthosilicate sol–gel matrices to produce a PtNHase:sol–gel biomaterial. The PtNHase:sol–gel biomaterial catalyzed the conversion of 600 mM acrylonitrile to acrylamide in 60 min at 35 °C with a yields of >90%. Treatment of the biomaterial with proteases confirmed that the catalytic activity is due to the encapsulated enzyme and not surface bound NHase. The biomaterial retained 50% of its activity after being used for a total of 13 consecutive reactions for the conversion of acrylonitrile to acrylamide. The thermostability and long-term storage of the PtNHase:sol–gel …

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A New Colorimetric Assay For Methionyl Aminopeptidases: Examination Of The Binding Of A New Class Of Pseudopeptide Analog Inhibitors, Sanghamitra Mitra, Anna Dygas-Holz, Jiri Jiracek, Miroslava Zertova, Lenka Zakova, Richard Holz Mar 2015

A New Colorimetric Assay For Methionyl Aminopeptidases: Examination Of The Binding Of A New Class Of Pseudopeptide Analog Inhibitors, Sanghamitra Mitra, Anna Dygas-Holz, Jiri Jiracek, Miroslava Zertova, Lenka Zakova, Richard Holz

Richard C. Holz

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (l-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV–Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using l-Met-p-NA, the kinetic constants kcat and Km were determined for EcMetAP-I and PfMetAP-II …

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Mechanistic Studies On The Aminopeptidase From Aeromonas Proteolytica:  A Two-Metal Ion Mechanism For Peptide Hydrolysis, Guanjing Chen, Tanya Edwards, Ventris D'Souza, Richard Holz Mar 2015

Mechanistic Studies On The Aminopeptidase From Aeromonas Proteolytica:  A Two-Metal Ion Mechanism For Peptide Hydrolysis, Guanjing Chen, Tanya Edwards, Ventris D'Souza, Richard Holz

Richard C. Holz

The aminopeptidase from Aeromonas proteolytica (AAP) is uncompetitively inhibited by fluoride ion at pH 8.0 with an inhibition constant (Ki) of 30 mM. Thus, fluoride inactivates AAP only after substrate binding, and only a single fluoride ion binds to AAP. On the other hand, chloride ion does not inhibit AAP up to concentrations of 2 M at pH 8.0. The pH dependence of fluoride inhibition of AAP was measured over the pH range 6.0−9.5. Between pH values of 6.0 and 9.0, fluoride ion acts as a pure uncompetitive inhibitor of AAP, and the Ki increases from …

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Spectroscopic Characterization Of The Europium(Iii) Complexes Of A Series Of N,N'-Bis(Carboxymethyl) Macrocyclic Ether Bis(Lactones), Richard Holz, C. Chang, William Horrocks Mar 2015

Spectroscopic Characterization Of The Europium(Iii) Complexes Of A Series Of N,N'-Bis(Carboxymethyl) Macrocyclic Ether Bis(Lactones), Richard Holz, C. Chang, William Horrocks

Richard C. Holz

The Eu3+ and Y3+ complexes of a series of N,N'-bis(carboxymethyl) macrocyclic ether bis(lactone) ligands derived from ethyl-enediaminetetraacetic acid (EDTA) were characterized in solution by using Eu3+ laser-induced luminescence and 1H and 13CNMR spectroscopy. The complexation of EDTA was also studied for comparison purposes. These ligands form 1:1 complexes with Eu3+ at the concentrations studied (10 µM), with the luminescence lifetimes in H2O and D2O providing the number of coordinated water molecules. The 7F0 -> 5D0 excitation spectra indicate that for each of …

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Epr And X-Ray Crystallographic Characterization Of The Product-Bound Form Of The MnIi-Loaded Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja Copik, Boguslaw Nocek, Sabina Swierczek, Shane Ruebush, Se Jang, Lu Meng, Ventris D'Souza, John Peters, Brian Bennett, Richard Holz Mar 2015

Epr And X-Ray Crystallographic Characterization Of The Product-Bound Form Of The MnIi-Loaded Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja Copik, Boguslaw Nocek, Sabina Swierczek, Shane Ruebush, Se Jang, Lu Meng, Ventris D'Souza, John Peters, Brian Bennett, Richard Holz

Richard C. Holz

Methionine aminopeptidases (MetAPs) are ubiquitous metallohydrolases that remove the N-terminal methionine from nascent polypeptide chains. Although various crystal structures of MetAP in the presence of inhibitors have been solved, the structural aspects of the product-bound step has received little attention. Both perpendicular- and parallel-mode electron paramagnetic resonance (EPR) spectra were recorded for the MnII-loaded forms of the type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs in the presence of the reaction product l-methionine (l-Met). In general, similar EPR features were observed for both [MnMn(EcMetAP-I)]−l-Met and [MnMn(PfMetAP-II)]−l-Met. The observed perpendicular-mode EPR spectra consisted of a six-line hyperfine pattern at g = …

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One- And Two-Dimensional Proton Nmr Studies Of The Active Site Of Iron(Ii) Superoxide Dismutase From Escherichia Coli, Li-June Ming, John Lynch, Richard Holz, Lawrence Que Mar 2015

One- And Two-Dimensional Proton Nmr Studies Of The Active Site Of Iron(Ii) Superoxide Dismutase From Escherichia Coli, Li-June Ming, John Lynch, Richard Holz, Lawrence Que

Richard C. Holz

The iron(II) superoxide dismutase (FeSOD) from Escherichia coli exhibits relatively sharp well-resolved paramagnetically shifted NMR signals. These signals can be associated with the endogenous ligands characterized by X-ray crystallography for the oxidized form FeIlISOD. Our results demonstrate that the active site remains intact upon reduction of the Fe(III) site, retaining the same coordination modes for the three N,-coordinated His residues (presumably His-26, His-75, and His-162) and the Asp residue (presumably Asp-158). The N-H resonances of the coordinated histidines are found at 88, 43, and 37 ppm, while the signals at 24.5 (E), 19 (G), and 15 (J) ppm …

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Kinetic, Spectroscopic, And X-Ray Crystallographic Characterization Of The Functional E151h Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Aaron Moulin, Sabina Swierczek, Dagmar Ringe, Gregory Petsko, Brian Bennett, Richard Holz Mar 2015

Kinetic, Spectroscopic, And X-Ray Crystallographic Characterization Of The Functional E151h Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Aaron Moulin, Sabina Swierczek, Dagmar Ringe, Gregory Petsko, Brian Bennett, Richard Holz

Richard C. Holz

Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the d-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in …

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Electrochemical Properties Of The Diiron Core Of Uteroferrin And Its Anion Complexes, Dan Wang, Richard Holz, Sheila David, Lawrence Que, Marian Stankovich Mar 2015

Electrochemical Properties Of The Diiron Core Of Uteroferrin And Its Anion Complexes, Dan Wang, Richard Holz, Sheila David, Lawrence Que, Marian Stankovich

Richard C. Holz

The reduction potentials (Em) of the purple acid phosphatase from porcine uterus, uteroferrin (Uf), and its phosphate, arsenate, and molybdate complexes were determined by coulometric methods at various pH values. The midpoint potential of Uf at the pH value for optimal enzyme activity (pH 5) was found to be +367 mV versus a normal hydrogen electrode (NHE), while at pH 6.01 Uf exhibits a reduction potential of +306 mV. At pH 6.01 molybdate was found to shift the potential of Uf more positive by 192 mV, while phosphate and arsenate shift the potential of Uf more negative by 193 and …

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Spectroscopic And Thermodynamic Characterization Of The E151d And E151a Altered Leucine Aminopeptidases From Aeromonas Proteolytica, Krzysztof Bzymek, Sabina Swierczek, Brian Bennett, Richard Holz Mar 2015

Spectroscopic And Thermodynamic Characterization Of The E151d And E151a Altered Leucine Aminopeptidases From Aeromonas Proteolytica, Krzysztof Bzymek, Sabina Swierczek, Brian Bennett, Richard Holz

Richard C. Holz

Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 Å in carboxypeptidase G2 and 3.30 and 3.63 Å in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by …

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Substrate Specificity, Metal Binding Properties, And Spectroscopic Characterization Of The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, David Bienvenue, Danuta Gilner, Ryan Davis, Brian Bennett, Richard Holz Mar 2015

Substrate Specificity, Metal Binding Properties, And Spectroscopic Characterization Of The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, David Bienvenue, Danuta Gilner, Ryan Davis, Brian Bennett, Richard Holz

Richard C. Holz

The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to ∼20% of the Zn(II)-loaded enzyme. The order of the observed kcat values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) ∼ Cu(II) ∼ Mg(II). DapE was shown to only hydrolyze l,l-N-succinyl-diaminopimelic acid (l,l-SDAP) and was inactive toward d,l-, l,d-, and d,d-SDAP. DapE was also inactive toward several acetylated amino acids as …

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Mono-N-Acyl-2,6-Diaminopimelic Acid Derivatives: Analysis By Electromigration And Spectroscopic Methods And Examination Of Enzyme Inhibitory Activity, Jan Hlaváček, Miloslava Vítovcová, Petra Sázelová, Jan Pícha, Václav Vaněk, Miloš Buděšínský, Jiří Jiráček, Danuta Gillner, Richard Holz, Ivan Mikšík, Václav Kašička Mar 2015

Mono-N-Acyl-2,6-Diaminopimelic Acid Derivatives: Analysis By Electromigration And Spectroscopic Methods And Examination Of Enzyme Inhibitory Activity, Jan Hlaváček, Miloslava Vítovcová, Petra Sázelová, Jan Pícha, Václav Vaněk, Miloš Buděšínský, Jiří Jiráček, Danuta Gillner, Richard Holz, Ivan Mikšík, Václav Kašička

Richard C. Holz

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary …

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Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Aliphatic Alcohols. Characterization Of The Hydrophobic Substrate Recognition Site, Leila Ustynyuk, Brian Bennett, Tanya Edwards, Richard Holz Mar 2015

Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Aliphatic Alcohols. Characterization Of The Hydrophobic Substrate Recognition Site, Leila Ustynyuk, Brian Bennett, Tanya Edwards, Richard Holz

Richard C. Holz

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, …

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Kinetic And Spectroscopic Characterization Of The H178a Methionyl Aminopeptidase From Escherichia Coli, Alicja Copik, Sabina Swierczek, W. Lowther, Ventris D'Souza, Brian Matthews, Richard Holz Mar 2015

Kinetic And Spectroscopic Characterization Of The H178a Methionyl Aminopeptidase From Escherichia Coli, Alicja Copik, Sabina Swierczek, W. Lowther, Ventris D'Souza, Brian Matthews, Richard Holz

Richard C. Holz

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. …

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Slow-Binding Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Peptide Thiols: Synthesis And Spectroscopic Characterization, Kristi Huntington, David Bienvenue, Yaoming Wei, Brian Bennett, Richard Holz, Dehua Pei Mar 2015

Slow-Binding Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Peptide Thiols: Synthesis And Spectroscopic Characterization, Kristi Huntington, David Bienvenue, Yaoming Wei, Brian Bennett, Richard Holz, Dehua Pei

Richard C. Holz

Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a−c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potencies (KI*) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin. The corresponding alcohols (2a−b) are simple competitive inhibitors of much lower potencies (KI = 23 and 360 μM). These data suggest that the free thiols are involved in the formation of the E·I and E·I* complexes, presumably serving as a metal ligand. To investigate the nature of the interaction …

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The Aminopeptidase From Aeromonas Proteolytica: Structure And Mechanism Of Co-Catalytic Metal Centers Involved In Peptide Hydrolysis, Richard Holz Mar 2015

The Aminopeptidase From Aeromonas Proteolytica: Structure And Mechanism Of Co-Catalytic Metal Centers Involved In Peptide Hydrolysis, Richard Holz

Richard C. Holz

Enzymes containing multi-metal active sites are central to numerous biological processes and, consequently, characterization of their structure and function is a problem of outstanding importance. One of the least-explored groups of enzymes is the hydrolases that contain dinuclear metal centers. These enzymes play key roles in carcinogenesis, tissue repair, and protein degradation processes. In addition, some of these enzymes can catalyze the hydrolysis of phosphorus(V) compounds found in nerve gases and agricultural neurotoxins. The determination of detailed reaction mechanisms for these enzymes is required for the design of highly potent, specific inhibitors that can function as potential pharmaceuticals. Hydrolytic enzymes …

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The Aminopeptidase From Aeromonas Proteolytica Can Function As An Esterase, David Bienvenue, Rebecca Matthew, Dagmar Ringe, Richard Holz Mar 2015

The Aminopeptidase From Aeromonas Proteolytica Can Function As An Esterase, David Bienvenue, Rebecca Matthew, Dagmar Ringe, Richard Holz

Richard C. Holz

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester (L-Leu-OEt) with a rate of 96±5 s–1 and a K m of 700 µM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67±5 s–1. The k cat values for the hydrolysis of L-Leu-OEt and L-leucine-p-nitroanilide (L-pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature …

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Characterization Of The Catalytically Active Mn(Ii)-Loaded Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade Mcgregor, Sabina Swierczek, Brian Bennett, Richard Holz Mar 2015

Characterization Of The Catalytically Active Mn(Ii)-Loaded Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade Mcgregor, Sabina Swierczek, Brian Bennett, Richard Holz

Richard C. Holz

The catalytically competent Mn(II)-loaded form of the argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli (ArgE) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N-acetyl-l-ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k cat and K m values of 550 s−1 and 0.8 mM, respectively, providing a catalytic efficiency (k cat/K m) of 6.9 × 105 M−1 s−1. The ArgE dissociation constant (K d) for Mn(II) was determined to be 0.18 μM, correlating well with a value obtained by isothermal titration …

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