argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli contains a dinuclear metalloactive site.
The catalytic and structural properties of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by approximately 20 times. Moreover, ArgE exhibits approximately 90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-L-ornithine ( NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 degrees C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co x (ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin-spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2).[1]References
- argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli contains a dinuclear metalloactive site. McGregor, W.C., Swierczek, S.I., Bennett, B., Holz, R.C. J. Am. Chem. Soc. (2005) [Pubmed]
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