Dissertation/Thesis Abstract

Mechanistic studies on the methionine aminopeptidase and peptide deformylase catalyzed reactions
by Sule, Nitesh Vivekananda, Ph.D., North Dakota State University, 2013, 273; 3603988
Abstract (Summary)

In the search for novel antibiotic and therapeutic targets, methionine aminopeptidase (MetAP) and peptide deformylase (PDF) have recently been identified as eminently compelling. These enzymes are involved in the co-translational modification of nascent polypeptides, affecting majority of the cellular proteome across all the kingdoms of living organisms. As a result, the various isoforms of MetAP and PDF have been successfully targeted by antimalarial and anti-cancer drugs. However, in spite of great interest in the bacterial forms of these enzymes as potent antibiotic targets, efforts to develop such agents have failed. This investigation is a study of the biochemical and biophysical features of the E. coli isoforms of MetAP and PDF in order to understand the unique characteristics of these bacterial enzymes.

The catalytic properties of these enzymes were studied using a combination of direct and coupled spectrometric assays. Potent inhibitors of MetAP were identified by screening a focused library of compounds and potential pharmacophores were determined. The inhibitor-enzyme interactions were further studied via steady-state and transient kinetic methods. While attempting to enhance the solubility of the tight-binding inhibitors using cyclodextrins, a novel substrate-driven mode of enzyme inhibition by 2-hydroxypropyl-β-cyclodextrin was discovered. The metal-ion binding iii properties of MetAP were studied and in this effort, the luminescence of the trivalent lanthanide ion europium was identified as a convenient signal for monitoring metal- ion binding to the enzyme. Moreover, europium was found to catalytically activate MetAP. These properties allowed the characterization of MetAP—metal-ion binding with various metals, and this represents the most comprehensive study of the MetAP metal-ion interactions. The C-terminal domain of PDF is implicated in imparting highly unusual properties to PDF, hence the stability of PDF was characterized with respect to this domain. The truncated form of PDF lacking the C-terminal domain was found to be remarkably robust with the secondary structure as well as catalytic activity being very resistant to loss by heat. The evidence suggests additional roles for the C-terminal domain in regulating PDF. Overall, the work described here provides new understanding and avenues for enzymological pursuits of MetAP, PDF and similar valuable targets.

Indexing (document details)
Advisor: Srivastava, DK
Commitee: Liu, Guodong, Pruess, Birgit, Sun, Wenfang
School: North Dakota State University
Department: Chemistry and Biochemistry
School Location: United States -- North Dakota
Source: DAI-B 75/03(E), Dissertation Abstracts International
Subjects: Biochemistry, Biophysics
Keywords: Catalysis, Inhibitors, Metal-ions, Methionine aminopeptidase, Peptide deformylase
Publication Number: 3603988
ISBN: 978-1-303-58961-4
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