Title

Characterization of the Catalytically Active Mn(II)-loaded argE-encoded N-acetyl-L-ornithine Deacetylase from Escherichia coli

Document Type

Article

Language

eng

Format of Original

11 p.

Publication Date

6-2007

Publisher

Springer

Source Publication

Journal of Biological Inorganic Chemistry

Source ISSN

0949-8257

Original Item ID

doi: 10.1007/s00775-007-0211-4

Abstract

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 calorimetry of 0.30 μM for the first metal binding event and 5.3 μM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded ArgE was linear from 15 to 55 °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 ΔG = 58.1 kJ mol−1, ΔH = 47.7 kJ mol−1, and ΔS = –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 μM. 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 µ-alkoxide bridge between the two metal ions.

Comments

Journal of Biological Inorganic Chemistry, Vol. 12, No. 5 (June 2007): 603-613. DOI.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.

Richard Holz was affiliated with Loyola University-Chicago and Utah State University at the time of publication.