title: Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay.
abstract: A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate. Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore. The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion. The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors. These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor. This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.
authors.affiliation: Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA.
authors.forename: Kiet T
authors.initials: KT
authors.lastname: Nguyen
authors.forename: Xubo
authors.initials: X
authors.lastname: Hu
authors.forename: Dehua
authors.initials: D
authors.lastname: Pei
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D040761:Biomimetic Materials / Q000737:chemistry; D020789:Cathepsin C / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D010456:Peptides, Cyclic / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010456:Peptides, Cyclic; D020789:Cathepsin C; D000581:Amidohydrolases; C084507:peptide deformylase