A direct spectrophotometric assay for peptide deformylase.
A direct UV-VIS spectrophotometric assay has been developed for peptide deformylase. This assay employs a novel class of peptide mimetics as deformylase substrates which, upon enzymatic removal of the N-terminal formyl group, rapidly release free thiols. The released thiols are quantitated using Ellman's reagent. A variety of peptide analogues that contain beta-thiaphenylalanine or beta-thiamethionine as the N-terminal residue were synthesized and found to be excellent substrates of the peptide deformylase from Escherichia coli (k(cat)/K(M) = 6.9 x 10(5) M(-1) s(-1) for the most reactive substrate). The deformylase reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion. The versatility of the assay has been demonstrated by its application to kinetic characterization of the deformylase, pH profile studies, and enzyme inhibition assays. The assay can also be performed in an end-point fashion. The results demonstrate that this assay is a simple, highly sensitive, and rapid method to study kinetic properties of deformylases without the use of any coupling enzymes.
Guo
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Department of Chemistry, Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Ravi Rajagopalan
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Pei
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D000581:Amidohydrolases; D000626:Aminopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D009842:Oligopeptides; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase