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Full-Text Articles in Physical Sciences and Mathematics
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
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 …
Kinetic And Spectroscopic Characterization Of The E134a- And E134d-Altered Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, Ryan Davis, David Bienvenue, Sabina Swierczek, Danuta Gilner, Lakshman Rajagopal, Brian Bennett, Richard Holz
Kinetic And Spectroscopic Characterization Of The E134a- And E134d-Altered Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, Ryan Davis, David Bienvenue, Sabina Swierczek, Danuta Gilner, Lakshman Rajagopal, Brian Bennett, Richard Holz
Richard C. Holz
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% activity), aspartate (0.09%), and alanine …
The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae Contains Two Active-Site Histidine Residues, Danuta Gillner, David L. Bienvenue, Boguslaw P. Nocek, Andzrej Joachimiak, Vincentos Zachary, Brian Bennett, Richard C. Holz
The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae Contains Two Active-Site Histidine Residues, Danuta Gillner, David L. Bienvenue, Boguslaw P. Nocek, Andzrej Joachimiak, Vincentos Zachary, Brian Bennett, Richard C. Holz
Richard C. Holz
The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the …