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Structural and Functional Characterization of Aminopeptidase N (PEPN) from Escherichia coli

Golich, Frank Carl
http://rave.ohiolink.edu/etdc/view?acc_num=miami1143229893

Abstract Details

2006, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
Aminopeptidases are ubiquitous hydrolytic enzymes that hydrolyze the N-terminal amino acids from peptides/proteins. Aminopeptidase N (PepN) primarily cleaves basic and hydrophobic residues from peptides. PepN in mammals is membrane-associated and has been implicated in viral infections and in tumor cell invasion. Bacterial PepNs are cytosolic, and E. coli PepN is the sole alanyl aminopeptidase in this organism. Bacterial proteinases have been suggested to be potential targets for the generation of novel antibiotics. Before inhibitor design efforts can be made, the structural and mechanistic characterization of the target is required. Escherichia coli PepN was cloned into pET26b, over-expressed in E. coli, and purified using Q-Sepharose chromatography. This protocol yielded over 17 mg of purified, recombinant PepN per liter of culture. Gel filtration chromatography revealed that PepN exists as a monomer. MALDI-TOF mass spectra showed that the enzyme has a molecular mass ca. 99 kDa. Metal analyses demonstrated that as-isolated, recombinant PepN binds 0.5 and <0.1 equivalents of iron and zinc, respectively. The addition of Zn(II) to recombinant PepN inhibits catalytic activity, while the addition of iron causes little change in activity. Further metal binding studies revealed that recombinant PepN tightly binds 5 equivalents of iron and < 0.1 equivalents of Zn(II). CD and fluorescence studies revealed that iron is not involved in the structure of PepN. EPR, NMR, and UV-Vis studies revealed that one of the irons binds at a site containing 2 histidines and at least 1 cysteine as ligands. Chemical modification studies suggest that cysteine is involved in the catalytic mechanism. pH Dependence and solvent isotope studies indicate several rate-limiting proton transfers, which can be tentatively assigned to cysteine and histidine residues. Stopped-flow kinetic studies, along with kinetic simulations, demonstrate that PepN utilizes a mechanism with ordered release of products and a rate-determining chemistry step. The first reaction mechanism for E. coli PepN is proposed. The results of this work can be used to guide future inhibitor design efforts, and these inhibitors may prove to be novel antibiotics.
Michael Crowder (Advisor)
119 p.
Aminopeptidase; PepN; Aminopeptidase N; E. coli; kinetics; L-ala-p-nitroanilide

Recommended Citations

Citations

  • Golich, F. C. (2006). Structural and Functional Characterization of Aminopeptidase N (PEPN) from Escherichia coli [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1143229893

    APA Style (7th edition)

  • Golich, Frank. Structural and Functional Characterization of Aminopeptidase N (PEPN) from Escherichia coli. 2006. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1143229893.

    MLA Style (8th edition)

  • Golich, Frank. "Structural and Functional Characterization of Aminopeptidase N (PEPN) from Escherichia coli." Doctoral dissertation, Miami University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=miami1143229893

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Miami University and OhioLINK.