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Full-Text Articles in Physics
Low Temperature Photo-Oxidation Of Chloroperoxidase Compound Ii, Xinting Yuan, Xin Sheng, John A. Horner, Brian Bennett, Leslie W. -M. Fung, Martin Newcomb
Low Temperature Photo-Oxidation Of Chloroperoxidase Compound Ii, Xinting Yuan, Xin Sheng, John A. Horner, Brian Bennett, Leslie W. -M. Fung, Martin Newcomb
Physics Faculty Research and Publications
Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as − 50 °C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene. EPR spectra at 77 K gave the amount of ferric protein at each stage in the reaction sequence. The PN oxidation step …
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000-fold decrease in kca towards …
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% …
Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By L-Leucinethiol: Kinetic And Spectroscopic Characterization Of A Slow, Tight-Binding Inhibitor–Enzyme Complex, David L. Bienvenue, Brian Bennett, Richard C. Holz
Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By L-Leucinethiol: Kinetic And Spectroscopic Characterization Of A Slow, Tight-Binding Inhibitor–Enzyme Complex, David L. Bienvenue, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
The peptide inhibitor l-leucinethiol (LeuSH) was found to be a potent, slow-binding inhibitor of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potency (KI*) of LeuSH was 7 nM while the corresponding alcohol l-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency (KI=17 μM). These data suggest that the free thiol is likely involved in the formation of the E·I and E·I* complexes, presumably providing a metal ligand. In order to probe the nature of the interaction of LeuSH and LeuOH with the dinuclear active site of AAP, we have …