Document Type
Article
Language
eng
Format of Original
10 p.
Publication Date
4-2000
Publisher
American Chemical Society
Source Publication
Biochemistry
Source ISSN
0006-2960
Original Item ID
doi: 10.1021/bi9925827
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.
Recommended Citation
D'Souza, Ventris M.; Bennett, Brian; Copik, Alicja J.; and Holz, Richard C., "Divalent Metal Binding Properties of the Methionyl Aminopeptidase from Escherichia coli" (2000). Physics Faculty Research and Publications. 43.
https://epublications.marquette.edu/physics_fac/43
Comments
Accepted version. Biochemistry, Vol. 39, No. 13 (April 2000): 3817-3826. DOI. © 2000 American Chemical Society. Used with permission.
Brian Bennett and Richard Holz were affiliated with Utah State University at the time of publication.