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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

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Abstract

EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~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≈2 EPR signal of mononuclear Mn(II) with Aav(55Mn)=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.

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Abbreviations

AMPP:

aminopeptidase P from E. coli

EcMetAP-I:

methionine aminopeptidase from E. coli

ZFS:

zero field splitting

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Acknowledgements

This work was supported by the National Institutes of Health (GM-56495 to R.C.H. and AI-056231 to B.B.) and the National Science Foundation (CHE-0240810 to R.C.H.). The Bruker ESP-300E EPR and ARX-400 NMR spectrometers were purchased with funds provided by the National Science Foundation (BIR-9413530 and CHE-9311730, respectively) and Utah State University. The methionyl aminopeptidase from E. coli was purified from a stock culture kindly provided by Drs Brian Matthews and W. Todd Lowther. The program EPRSim XOP was made available by Dr John Boswell, Oregon Graduate Institute.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Utah State University, Logan, UT , 84322-0300, USA

    Ventris M. D’souza, Robert S. Brown & Richard C. Holz

  2. Department of Biophysics, The Medical College of Wisconsin, Milwaukee, WI , 53226-0509, USA

    Brian Bennett

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  1. Ventris M. D’souza
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  2. Robert S. Brown
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  3. Brian Bennett
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  4. Richard C. Holz
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Correspondence to Richard C. Holz.

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D’souza, V.M., Brown, R.S., Bennett, B. et al. 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. J Biol Inorg Chem 10, 41–50 (2005). https://doi.org/10.1007/s00775-004-0611-7

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  • DOI: https://doi.org/10.1007/s00775-004-0611-7

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