Characterization of the active site and insight into the binding mode of the anti-angiogenesis agent fumagillin to the manganese(II)-loaded methionyl aminopeptidase from Escherichia coli.
EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (approximately 2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g approximately 2 EPR signal of mononuclear Mn(II) with A(av)((55)Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen atoms. Other features in the spectrum were observed only as the temperature was raised from that of liquid helium. The temperature dependences of these features are consistent with their assignment to excited state transitions in the S=1, 2 ... 5 non-Kramer's doublets, due to two antiferromagnetically coupled Mn(II) ions with an S=0 ground state. This assignment is supported by the observation of a characteristic 4.5 mT hyperfine pattern, and by the presence of signals in the parallel mode consistent with a non-Kramers' spin ladder. Upon the addition of the anti-angiogenesis agent fumagillin to [MnMn(EcMetAP-I)], very small changes were observed in the EPR spectrum. MALDI-TOF mass spectrometry indicated that fumagillin was, however, covalently coordinated to EcMetAP-I. Therefore, the inhibitory action of this anti-angiogenesis agent on EcMetAP-I appears to involve covalent binding to a polypeptide component at or near the active site rather than direct binding to the metal ions.
D'souza
Ventris M
VM
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA.
Brown
Robert S
RS
Bennett
Brian
B
Holz
Richard C
RC
D000626:Aminopeptidases / Q000378:metabolism*; D020533:Angiogenesis Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites; D003510:Cyclohexanes; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism* / Q000494:pharmacology*; D008345:Manganese / Q000378:metabolism* / Q000494:pharmacology; D063208:Methionyl Aminopeptidases; D008968:Molecular Conformation; D012717:Sesquiterpenes
D020533:Angiogenesis Inhibitors; D003510:Cyclohexanes; D005231:Fatty Acids, Unsaturated; D012717:Sesquiterpenes; D008345:Manganese; C026211:fumagillin; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases