title: Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli.
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 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 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 ( approximately 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. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O 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.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
authors.forename: V M
authors.initials: VM
authors.lastname: D'souza
authors.forename: B
authors.initials: B
authors.lastname: Bennett
authors.forename: A J
authors.initials: AJ
authors.lastname: Copik
authors.forename: R C
authors.initials: RC
authors.lastname: Holz
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry* / Q000378:metabolism*; D063208:Methionyl Aminopeptidases; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002413:Cations, Divalent; D003035:Cobalt; D007501:Iron; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases