Structural evidence that the methionyl aminopeptidase from Escherichia coli is a mononuclear metalloprotease.
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.
Cosper
N J
NJ
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
D'souza
V M
VM
Scott
R A
RA
Holz
R C
RC
D015502:Absorptiometry, Photon; D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D020533:Angiogenesis Inhibitors; D007093:Imidazoles; D011994:Recombinant Proteins; D003035:Cobalt; D006639:Histidine; D007501:Iron; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D008666:Metalloendopeptidases