Open Access. Powered by Scholars. Published by Universities.®
- Keyword
Articles 1 - 11 of 11
Full-Text Articles in Physics
Structural Studies On A Mitochondrial Glyoxalase Ii, Gishanthi P. K. Marasinghe, Ian M. Sander, Brian Bennett, Gopal R. Periyannan, Ke-Wu Yang, Christopher A. Makaroff, Michael W. Crowder
Structural Studies On A Mitochondrial Glyoxalase Ii, Gishanthi P. K. Marasinghe, Ian M. Sander, Brian Bennett, Gopal R. Periyannan, Ke-Wu Yang, Christopher A. Makaroff, Michael W. Crowder
Physics Faculty Research and Publications
Glyoxalase 2 is a β-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and 1H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 ± 0.15 eq of iron and 1.31 ± 0.05 eq of Zn(II) and to exhibit kcat and …
Spectroscopic And Thermodynamic Characterization Of The E151d And E151a Altered Leucine Aminopeptidases From Aeromonas Proteolytica, Krzysztof P. Bzymek, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Spectroscopic And Thermodynamic Characterization Of The E151d And E151a Altered Leucine Aminopeptidases From Aeromonas Proteolytica, Krzysztof P. Bzymek, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
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 …
Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli Contains A Dinuclear Metalloactive Site, Wade C. Mcgregor, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli Contains A Dinuclear Metalloactive Site, Wade C. Mcgregor, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
The catalytic and structural properties of the argE-encoded N-acetyl-l-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by ∼20 times. Moreover, ArgE exhibits ∼90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-l-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from …
The Octarepeat Domain Of The Prion Protein Binds Cu(Ii) With Three Distinct Coordination Modes At Ph 7.4, Madhuri Chattopadhyay, Eric D. Walter, Dustin J. Newell, Pilgrim J. Jackson, Eliah Aronoff-Spencer, Jack Peisach, Gary J. Gerfen, Brian Bennett, William E. Antholine, Glenn L. Millhauser
The Octarepeat Domain Of The Prion Protein Binds Cu(Ii) With Three Distinct Coordination Modes At Ph 7.4, Madhuri Chattopadhyay, Eric D. Walter, Dustin J. Newell, Pilgrim J. Jackson, Eliah Aronoff-Spencer, Jack Peisach, Gary J. Gerfen, Brian Bennett, William E. Antholine, Glenn L. Millhauser
Physics Faculty Research and Publications
The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, …
Kinetic, Spectroscopic, And X-Ray Crystallographic Characterization Of The Functional E151h Aminopeptidase From Aeromonas Proteolytica, Krzysztof P. Bzymek, Aaron Moulin, Sabina I. Swierczek, Dagmar Ringe, Gregory A. Petsko, Brian Bennett, Richard C. Holz
Kinetic, Spectroscopic, And X-Ray Crystallographic Characterization Of The Functional E151h Aminopeptidase From Aeromonas Proteolytica, Krzysztof P. Bzymek, Aaron Moulin, Sabina I. Swierczek, Dagmar Ringe, Gregory A. Petsko, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
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 …
Spectroscopic Studies On Cobalt(Ii)-Substituted Metallo-Β-Lactamase Imis From Aeromonas Veronii Bv. Sobria, Patrick A. Crawford, Ke-Wu Yang, Narayan Sharma, Brian Bennett, Michael W. Crowder
Spectroscopic Studies On Cobalt(Ii)-Substituted Metallo-Β-Lactamase Imis From Aeromonas Veronii Bv. Sobria, Patrick A. Crawford, Ke-Wu Yang, Narayan Sharma, Brian Bennett, Michael W. Crowder
Physics Faculty Research and Publications
In an effort to probe the structure of a group Bb metallo-β-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)1-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)1-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)1-ImiS. 1H NMR studies of Co(II)1-ImiS suggest that one histidine, one aspartic acid, and one cysteine …
A Five-Coordinate Metal Center In Co(Ii)-Substituted Vanx, Robert M. Breece, Alison L. Costello, Brian Bennett, Tara K. Sigdel, Megan L. Matthews, David L. Tierney, Michael W. Crowder
A Five-Coordinate Metal Center In Co(Ii)-Substituted Vanx, Robert M. Breece, Alison L. Costello, Brian Bennett, Tara K. Sigdel, Megan L. Matthews, David L. Tierney, Michael W. Crowder
Physics Faculty Research and Publications
In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of ∼100 m–1 cm–1, which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M = |±½〉, E/D = 0.14, greal(x,y) = 2.37, and grealS(z) = 2.03. These parameters …
Both Nucleophile And Substrate Bind To The Catalytic Fe(Ii)-Center In The Type-Ii Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja J. Copik, Sarah Waterson, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Both Nucleophile And Substrate Bind To The Catalytic Fe(Ii)-Center In The Type-Ii Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja J. Copik, Sarah Waterson, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
Metalloproteases utilize their active site divalent metal ions to generate a nucleophilic water/hydroxide. For methionine aminopeptidases (MetAPs), the exact location of this nucleophile, as well as of the substrate, with respect to the active site metal ion is unknown. In order to address this issue, we have examined the catalytically competent Fe(II)-loaded form of PfMetAP-II ([Fe(PfMetAP-II)]) in the absence and presence of both nitric oxide (NO) and the substrate-analogue inhibitor butaneboronic acid (BuBA) by kinetic and spectroscopic (EPR, UV−vis) methods. NO binds to [Fe(PfMetAP−II)] with a Kd of 200 μM forming an {FeNO}7 …
Direct Evidence That The Reaction Intermediate Of Metallo-Β-Lactamase L1 Is Metal Bound, James D. Garrity, Brian Bennett, Michael W. Crowder
Direct Evidence That The Reaction Intermediate Of Metallo-Β-Lactamase L1 Is Metal Bound, James D. Garrity, Brian Bennett, Michael W. Crowder
Physics Faculty Research and Publications
In an effort to probe the structure of the reaction intermediate of metallo-β-lactamase L1 when reacted with nitrocefin and other β-lactams, time-dependent absorption and rapid-freeze-quench (RFQ) EPR spectra were obtained using the Co(II)-substituted form of the enzyme. When using nitrocefin as the substrate, time-dependent absorption spectra demonstrate that Co(II)-substituted L1 utilizes a reaction mechanism, similar to that of the native Zn(II) enzyme, in which a short-lived intermediate forms. RFQ-EPR spectra of this intermediate demonstrate that the binding of substrate results in a change in the electronic properties of one or both of the Co(II)'s in the enzyme …
Epr And X-Ray Crystallographic Characterization Of The Product-Bound Form Of The MnIi-Loaded Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja J. Copik, Boguslaw P. Nocek, Sabina I. Swierczek, Shane Ruebush, Se Bok Jang, Lu Meng, Ventris M. D'Souza, John W. Peters, Brian Bennett, Richard C. Holz
Epr And X-Ray Crystallographic Characterization Of The Product-Bound Form Of The MnIi-Loaded Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja J. Copik, Boguslaw P. Nocek, Sabina I. Swierczek, Shane Ruebush, Se Bok Jang, Lu Meng, Ventris M. D'Souza, John W. Peters, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
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 …
Characterization Of The Active Site And Insight Into The Binding Mode Of The Anti-Angiogenesis Agent Fumagillin To The Manganese(Ii)-Loaded Methionyl Aminopeptidase From Escherichia Coli, Ventris M. D'Souza, Robert S. Brown, Brian Bennett, Richard C. Holz
Characterization Of The Active Site And Insight Into The Binding Mode Of The Anti-Angiogenesis Agent Fumagillin To The Manganese(Ii)-Loaded Methionyl Aminopeptidase From Escherichia Coli, Ventris M. D'Souza, Robert S. Brown, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g≈2 EPR signal of mononuclear Mn(II) with A av(55Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen …