Mechanism of Time-Dependent Inhibition of Polypeptide Deformylase by Actinonin
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- 作者：Glenn S. Van Aller ; Ravi Nandigama ; Chantal M. Petit ; Walt E. DeWolf ; Jr. ; Chad J. Quinn ; Kelly M. Aubart ; Magdalena Zalacain ; Siegfried B. Christensen ; Robert A. Copeland ; and Zhihong Lai
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：January 11, 2005
- 年：2005
- 卷：44
- 期：1
- 页码：253 - 260
- 全文大小：139K
- 年卷期：v.44,no.1(January 11, 2005)
- ISSN：1520-4995
文摘
Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for theremoval of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition ofbacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterizedusing steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observedunder steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism,in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremelyslow, but observable, off-rate (t1/2 for inhibitor dissociation 
s/entities/ge.gif">0.77 days). Stopped-flow measurement ofPDF fluorescence confirms formation of EI and provides a direct measurement of the association rate.Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upontightening of EI to form EI*, from Ki = 530 nM (EI) to 
subscribe/journals/bichaw/44/i01/eqn/bi048632be10001.gif"> s/entities/le.gif"> 0.23 nM (EI*). In sharp contrast, thepreviously reported small molecule PDF inhibitor, SB-543668, is a competitive, readily reversible inhibitor(t1/2 for dissociation = 2.8 s). In addition, we demonstrate that BB-3497 is also a time-dependent inhibitorof PDF with an extremely slow off-rate. The two-step inhibition model detailed herein for the inhibitionof Staphylococcus aureus PDF by actinonin and BB-3497 is consistent with a recent report on the time-dependent inhibition of Escherichia coli PDF by a macrocyclic peptidomimetic inhibitor [Ngugen, K. T.,et al. (2004) Bioorg. Chem. 32, 178-191]. This study substantially extends our understanding of PDFinhibition and may facilitate the development of novel antibiotics.