Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli
| Autor: | Sabina Swierczek, Richard C. Holz, Brian Bennett, Wade C. McGregor |
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| Rok vydání: | 2006 |
| Předmět: |
Metal ions in aqueous solution
Analytical chemistry chemistry.chemical_element Manganese Calorimetry Biochemistry Catalysis law.invention Amidohydrolases Inorganic Chemistry Apoenzymes law Leucine Escherichia coli Protease Inhibitors Enzyme Inhibitors Electron paramagnetic resonance Chemistry Hydrolysis Electron Spin Resonance Spectroscopy Substrate (chemistry) Isothermal titration calorimetry Arrhenius plot Dissociation constant Crystallography Kinetics Genes Bacterial Thermodynamics Spectrophotometry Ultraviolet |
| Zdroj: | Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 12(5) |
| ISSN: | 0949-8257 |
| Popis: | 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 x 10(5) M(-1) s(-1). The ArgE dissociation constant (K(d)) for Mn(II) was determined to be 0.18 microM, correlating well with a value obtained by isothermal titration calorimetry of 0.30 microM for the first metal binding event and 5.3 microM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded ArgE was linear from 15 to 55 degrees C, suggesting the rate-limiting step does not change as a function of temperature over this range. The activation energy, determined from the slope of this plot, was 50.3 kJ mol(-1). Other thermodynamic parameters were DeltaG(double dagger) = 58.1 kJ mol(-1), DeltaH(double dagger) = 47.7 kJ mol(-1), and DeltaS(double dagger) = -34.5 J mol(-1) K(-1). Similarly, plots of lnK(m) versus 1/T were linear, suggesting substrate binding is controlled by a single step. The natural product, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]leucine (bestatin), was found to be a competitive inhibitor of ArgE with a K (i) value of 67 muM. Electron paramagnetic resonance (EPR) data recorded for both [Mn(II)_(ArgE)] and [Mn(II)Mn(II)(ArgE)] indicate that the two Mn(II) ions form a dinuclear site. Moreover, the EPR spectrum of [Mn(II)Mn(II)(ArgE)] in the presence of bestatin indicates that bestatin binds to ArgE but does not form a micro-alkoxide bridge between the two metal ions. |
| Databáze: | OpenAIRE |
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