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Full-Text Articles in Physics
A Cobalt-Containing Eukaryotic Nitrile Hydratase, Salette Martinez, Xinhang Yang, Brian Bennett, Richard C. Holz
A Cobalt-Containing Eukaryotic Nitrile Hydratase, Salette Martinez, Xinhang Yang, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ)2 homodimer in solution, analogous to the α2β2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) …
Analyzing The Binding Of Co(Ii)-Specific Inhibitors To The Methionyl Aminopeptidases From Escherichia Coli And Pyrococcus Furiosus, Sanghamitra Mitra, George Sheppard, Jieyi Wang, Brian Bennett, Richard C. Holz
Analyzing The Binding Of Co(Ii)-Specific Inhibitors To The Methionyl Aminopeptidases From Escherichia Coli And Pyrococcus Furiosus, Sanghamitra Mitra, George Sheppard, Jieyi Wang, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to …
X-Ray Crystallographic Characterization Of The Co(Ii)-Substituted Tris-Bound Form Of The Aminopeptidase From Aeromonas Proteolytica, Petra Munih, Aaron Moulin, Carin Stamper, Brian Bennett, Dagmar Ringe, Gregory A. Petsko, Richard C. Holz
X-Ray Crystallographic Characterization Of The Co(Ii)-Substituted Tris-Bound Form Of The Aminopeptidase From Aeromonas Proteolytica, Petra Munih, Aaron Moulin, Carin Stamper, Brian Bennett, Dagmar Ringe, Gregory A. Petsko, Richard C. Holz
Physics Faculty Research and Publications
The X-ray crystal structure of the Co(II)-loaded form of the aminopeptidase from Aeromonas proteolytica ([CoCo(AAP)]) was solved to 2.2 Å resolution. [CoCo(AAP)] folds into an α/β globular domain with a twisted β-sheet hydrophobic core sandwiched between α-helices, identical to [ZnZn(AAP)]. Co(II) binding to AAP does not introduce any major conformational changes to the overall protein structure and the amino acid residues ligated to the dicobalt(II) cluster in [CoCo(AAP)] are the same as those in the native Zn(II)-loaded structure with only minor perturbations in bond lengths. The Co(II)–Co(II) distance is 3.3 Å. Tris(hydroxymethyl)aminomethane (Tris) coordinates to the dinuclear Co(II) active site …
Characterization Of The Catalytically Active Mn(Ii)-Loaded Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Characterization Of The Catalytically Active Mn(Ii)-Loaded Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
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 …
Kinetic And Spectroscopic Characterization Of The E134a- And E134d-Altered Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, Ryan S. Davis, David L. Bienvenue, Sabina I. Swierczek, Danuta M. Gilner, Lakshman Rajagopal, Brian Bennett, Richard C. Holz
Kinetic And Spectroscopic Characterization Of The E134a- And E134d-Altered Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, Ryan S. Davis, David L. Bienvenue, Sabina I. Swierczek, Danuta M. Gilner, Lakshman Rajagopal, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
Glutamate-134 (E134) is proposed to act as the general acid/base during the hydrolysis reaction catalyzed by the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae. To date, no direct evidence has been reported for the role of E134 during catalytic turnover by DapE. In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue. The Michaelis constant (K m) does not change upon substitution with aspartate but the rate of the reaction changes drastically in the following order: glutamate (100% …