Mechanism of time-dependent inhibition of polypeptide deformylase by actinonin.
Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under 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 extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF 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 upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*<or= 0.23 nM (EI*). In sharp contrast, the previously reported small molecule PDF inhibitor, SB-543668, is a competitive, readily reversible inhibitor (t(1/2) for dissociation = 2.8 s). In addition, we demonstrate that BB-3497 is also a time-dependent inhibitor of PDF with an extremely slow off-rate. The two-step inhibition model detailed herein for the inhibition of 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 PDF inhibition and may facilitate the development of novel antibiotics.
Van Aller
Glenn S
GS
Enzymology and Mechanistic Pharmacology, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
Nandigama
Ravi
R
Petit
Chantal M
CM
DeWolf
Walt E
WE
Quinn
Chad J
CJ
Aubart
Kelly M
KM
Zalacain
Magdalena
M
Christensen
Siegfried B
SB
Copeland
Robert A
RA
Lai
Zhihong
Z
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D006877:Hydroxamic Acids / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000201:enzymology
D000900:Anti-Bacterial Agents; D006877:Hydroxamic Acids; D000581:Amidohydrolases; C084507:peptide deformylase; C010531:actinonin