PMID:11683640
| Citation |
Cosper, NJ, D'souza, VM, Scott, RA and Holz, RC (2001) Structural evidence that the methionyl aminopeptidase from Escherichia coli is a mononuclear metalloprotease. Biochemistry 40:13302-9 |
|---|---|
| Abstract |
The Co and Fe K-edge extended X-ray absorption fine structure (EXAFS) spectra of the methionyl aminopeptidase from Escherichia coli (EcMetAP) have been recorded in the presence of 1 and 2 equiv of either Co(II) or Fe(II) (i.e., [Co(II)_(EcMetAP)], [Co(II)Co(II)(EcMetAP)], [Fe(II)_(EcMetAP)], and [Fe(II)Fe(II)(EcMetAP)]). The Fourier transformed data of both [Co(II)_(EcMetAP)] and [Co(II)Co(II)(EcMetAP)] are dominated by a peak at ca. 2.05 A, which can be fit assuming 5 light atom (N,O) scatterers at 2.04 A. Attempts to include a Co-Co interaction (in the 2.4-4.0 A range) in the curve-fitting parameters were unsuccessful. Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye-Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f '). These data suggest that a dinuclear Co(II) center does not exist in EcMetAP and that the first Co atom is located in the histidine-ligated side of the active site. The EXAFS data obtained for [Fe(II)_(EcMetAP)] and [Fe(II)Fe(II)(EcMetAP)] indicate that Fe(II) binds to EcMetAP in a similar site to Co(II). Since no X-ray crystallographic data are available for any Fe(II)-substituted EcMetAP enzyme, these data provide the first glimpse at the Fe(II) active site of MetAP enzymes. In addition, the EXAFS data for [Co(II)Co(II)(EcMetAP)] incubated with the antiangiogenesis drug fumagillin are also presented. |
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| Keywords |
Absorptiometry, Photon; Aminopeptidases/chemistry; Aminopeptidases/isolation & purification; Angiogenesis Inhibitors/pharmacology; Binding Sites; Cobalt/chemistry; Cobalt/metabolism; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli/enzymology; Histidine/chemistry; Humans; Imidazoles/chemistry; Iron/chemistry; Iron/metabolism; Kinetics; Metalloendopeptidases/chemistry; Metalloendopeptidases/metabolism; Methionyl Aminopeptidases; Models, Chemical; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Spectroscopy, Fourier Transform Infrared |
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