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Article
Divalent Metal Binding Properties of the Methionyl Aminopeptidase from Escherichia coli
Biochemistry
  • Ventris M. D'Souza, Utah State University
  • Brian Bennett, Marquette University
  • Alicja J. Copik, Utah State University
  • Richard C. Holz, Marquette University
Document Type
Article
Language
eng
Format of Original
10 p.
Publication Date
4-1-2000
Publisher
American Chemical Society
Original Item ID
doi: 10.1021/bi9925827
Disciplines
Abstract
The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 ± 0.2 μM and 0.2 ± 0.2 μM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of ∼50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 ± 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion (∼5%) of the sample exhibited detectable spin−spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. 1H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H2O and D2O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed.
Comments

Biochemistry, Vol. 39, No. 13 (April 2000): 3817-3826. DOI.

Brian Bennett and Richard Holz were affiliated with Utah State University at the time of publication.

Citation Information
Ventris M. D'Souza, Brian Bennett, Alicja J. Copik and Richard C. Holz. "Divalent Metal Binding Properties of the Methionyl Aminopeptidase from Escherichia coli" Biochemistry (2000) ISSN: 0006-2960
Available at: http://works.bepress.com/richard_holz/34/