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

Authors

Wade C. McGregor, Utah State UniversityFollow
Sabina I. Swierczek, Utah State UniversityFollow
Brian Bennett, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow

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

Accepted version. Journal of Biological Inorganic Chemistry, Vol. 12, No. 5 (June 2007): 603-613. DOI. © 2007 Springer Nature Switzerland AG. Part of Springer Nature. Used with permission.

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.

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Recommended Citation

McGregor, Wade C.; Swierczek, Sabina I.; Bennett, Brian; and Holz, Richard C., "Characterization of the Catalytically Active Mn(II)-loaded argE-encoded N-acetyl-L-ornithine Deacetylase from Escherichia coli" (2007). Physics Faculty Research and Publications. 76.
https://epublications.marquette.edu/physics_fac/76