Inhibition and Thermodynamics of Escherichia coli Peptide Deformylase
hydrogen deuterium exchange
isothermal titration calorimetry
MetadataShow full item record
Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Biochemistry Honors Theses; 2007
Peptide deformylase (PDF) is a bacterial metalloenzyme responsible for cleaving the formyl group from nascent polypeptides, aiding in their maturation. Because it is essential for the survival of bacterial cells and does not appear to play a necessary role in humans, PDF is an attractive antibacterial target. Actinonin is a naturally occurring compound that acts as a substrate analog to bind PDF and inhibit the enzyme’s activity, therefore providing a potential template for developing new antibiotics. Described here are various dynamic features of PDF and the PDF-actinonin complex and thermodynamic properties of the binding event of actinonin to PDF. Isothermal titration calorimetry (ITC) experiments were performed to provide thermodynamic information (KA, ΔG, ΔH, and ΔS) about the binding of actinonin to PDF. First, because the identity of the bound divalent metal affects the catalytic activities of PDF, the thermodynamics of actinonin binding to Ni-, Co-, and Zn-PDF were explored. The association constant (KA) was found to be highest for Zn-PDF and similar for the Ni- and Co-bond protein. These data also showed a correlation between the efficiency of catalysis (kcat/KM) and the thermodynamics (ΔH, ΔS) of actinonin binding. To assess the degree to which changes in protein motions contribute to binding, the change in heat capacity (ΔCp) between the free and bound state was determined by measuring the temperature dependence of the enthalpy change, ΔH, upon binding. The effects of salt concentration and buffer on the ΔH were assessed to control for binding-linked processes that could contribute to the measured change in heat capacity. The value of ΔCp, -177.5 cal/mol∙K, is larger in magnitude than the value expected from changes in molecular surface hydration, consistent with the hypothesis that changes in molecular motion do contribute to the thermodynamics of actinonin binding. To characterize the effect of actinonin binding on the stability and motions in PDF, hydrogen-deuterium (H/D) exchange experiments using nuclear magnetic resonance (NMR) spectroscopy were performed on both free and actinonin-bound PDF. The binding of actinonin resulted in a large increase in protection of amide bonds in the binding pocket of PDF and entropically favored decreases of protection of amide bonds on the opposite side of the protein. These results are consistent with previous relaxation data.
Items in Knowledge Bank are protected by copyright, with all rights reserved, unless otherwise indicated.