Thee long range goals of this research are to define the reaction mechanism and substrate specificity of the cobalt(II)-dependent methionine aminopeptidase from Escherichia coli (MAP). In eukaryotic as well as in prokaryotic cells, methiomine aminopeptidases selectively cleave methionine residues from the N-termini of terminal polypeptide chains. In the cytosol of eukaryotes, all proteins are initiated with an N-terminal methionine. The composition of mature N-termini play important roles in the directed degradation and cellular targeting of proteins involved in signal transduction, protein trafficking, cancer cell growth, and viral infection. Recently, one of the two MAP's found in yeast was shown to be the target of the angiogenic chemotherapeutic agents fumagilln and its analog AGM 1470. Therefore, MAP's appear to play a critical role in the proliferation of endothelial cells and likely serve as important targets for inhibiting the growth and proliferation of tumors. As isolated from several bacterial and mammalian sources, MAPs are maximally stimulated by two g-atoms of Co(II). On the other hand, the addition of Zn(II) or Mg(II) to apo- MAP's do not provide active enzymes. The MAP from E. coli has been shown by X-ray crystallography to contain a dinuclear cobalt(II) active site. Therefore, MAP is a member of a new class of cobalt- containing enzymes that includes mammalian MAP' isolated from both porcine liver and humans. Our approach will be to utilize the X-ray crystal structures of the MAP's from E. coli and P. furiosus in conjunction with site mutagenesis, biochemical, and spectroscopic methods to gain fingerprints of each step in the catalytic mechanism. The MAP from E. coli is an ideal enzyme for a study of this type because it is small, highly soluble, readily available in large amounts, and is capable of being genetically manipulated.
The specific aims of this proposal are to: 1) define the structural and magnetic properties of the dicobalt(II) an diiron(II) MAP enzymes, 2) characterize how substrate analog inhibitors interact with both the dicobalt(II) and diiron(II) MAP enzymes, 3) determine whether the nucleophilic hydroxide moiety, a key element in the proposed peptide bond cleavage mechanism, is coordinated to one or both metal ions in MAP, 4) prepare and purify variant enzymes with altered active site carboxylic acid, histidine, threonine, and tyrosine residues, 5) provide evidence for and propose a detailed mechanism for MAP. It is anticipated that the successful completion of the studies described in this proposal will provide new insights into the hydrolytic reaction catalyzed by MAP and will ultimately assist in the design and synthesis of new chemotherapeutic agents targeted specifically to MAP's.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM056495-01A2
Application #
2759808
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1999-01-01
Project End
2002-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Utah State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Logan
State
UT
Country
United States
Zip Code
84322
Copik, Alicja J; Waterson, Sarah; Swierczek, Sabina I et al. (2005) Both nucleophile and substrate bind to the catalytic Fe(II)-center in the type-II methionyl aminopeptidase from Pyrococcus furiosus. Inorg Chem 44:1160-2
Copik, Alicja J; Nocek, Boguslaw P; Swierczek, Sabina I et al. (2005) EPR and X-ray crystallographic characterization of the product-bound form of the MnII-loaded methionyl aminopeptidase from Pyrococcus furiosus. Biochemistry 44:121-9
Swierczek, Krzysztof; Copik, Alicja J; Swierczek, Sabina I et al. (2005) Molecular discrimination of type-I over type-II methionyl aminopeptidases. Biochemistry 44:12049-56
D'souza, Ventris M; Brown, Robert S; Bennett, Brian et al. (2005) 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
Hu, Xubo; Zhu, Jinge; Srivathsan, Sumant et al. (2004) Peptidyl hydroxamic acids as methionine aminopeptidase inhibitors. Bioorg Med Chem Lett 14:77-9
Holz, Richard C; Bzymek, Krzysztof P; Swierczek, Sabina I (2003) Co-catalytic metallopeptidases as pharmaceutical targets. Curr Opin Chem Biol 7:197-206
Copik, Alicja J; Swierczek, Sabina I; Lowther, W Todd et al. (2003) Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli. Biochemistry 42:6283-92
Meng, Lu; Ruebush, Shane; D'souza, Ventris M et al. (2002) Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus. Biochemistry 41:7199-208
Bennett, Brian; Antholine, William E; D'souza, Ventris M et al. (2002) Structurally distinct active sites in the copper(II)-substituted aminopeptidases from Aeromonas proteolytica and Escherichia coli. J Am Chem Soc 124:13025-34
D'souza, Ventris M; Swierczek, Sabina I; Cosper, Nathaniel J et al. (2002) Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases. Biochemistry 41:13096-105

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