Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli.
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.
McGregor
Wade C
WC
The Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA.
Swierczek
Sabina I
SI
Bennett
Brian
B
Holz
Richard C
RC
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D001051:Apoenzymes / Q000378:metabolism; D002151:Calorimetry; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D006868:Hydrolysis; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D001051:Apoenzymes; D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; D008345:Manganese; D000581:Amidohydrolases; C018732:N-acetylornithine deacetylase; D007930:Leucine; C012211:ubenimex