Faculty Collaboration Database

Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli. J Biol Inorg Chem 2007 Jun;12(5):603-13

Date

03/03/2007

Pubmed ID

17333302

DOI

10.1007/s00775-007-0211-4

Scopus ID

2-s2.0-34249875410 (requires institutional sign-in at Scopus site)   9 Citations

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 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.

Author List

McGregor WC, Swierczek SI, Bennett B, Holz RC

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University

MESH terms used to index this publication - Major topics in bold

Amidohydrolases
Apoenzymes
Calorimetry
Catalysis
Electron Spin Resonance Spectroscopy
Enzyme Inhibitors
Escherichia coli
Genes, Bacterial
Hydrolysis
Kinetics
Leucine
Manganese
Protease Inhibitors
Spectrophotometry, Ultraviolet
Thermodynamics