A Direct Spectrophotometric Assay for Peptide Deformylase Xiao-Chuan Guo, 1 P. T. Ravi Rajagopalan, 1 and Dehua Pei 2 Department of Chemistry, Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210 Received May 19, 1999 A direct UV–VIS spectrophotometric assay has been developed for peptide deformylase. This assay em- ploys 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 re- agent. A variety of peptide analogues that contain -thiaphenylalanine or -thiamethionine as the N-ter- minal residue were synthesized and found to be excel- lent substrates of the peptide deformylase from Esch- erichia coli (k cat /K M  6.9  10 5 M 1 s 1 for the most reactive substrate). The deformylase reaction is con- veniently 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 stud- ies, 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 sensi- tive, and rapid method to study kinetic properties of deformylases without the use of any coupling en- zymes. © 1999 Academic Press Key Words: peptide deformylase; kinetics; assay; -thiaphenylalanine; Ellman’s reagent. Protein synthesis in prokaryotes initiates with an N-formylmethionyl-tRNA i , resulting in N-terminal formylation of all nascent polypeptides (1). Peptide deformylase (PDF) 3 catalyzes the subsequent removal of the formyl group from the majority of bacterial pro- teins (2– 4). Although the precise functions of the formylation and deformylation steps remain somewhat obscure, genetic studies have shown that the PDF ac- tivity is essential for bacterial survival (5, 6). Since PDF is apparently absent in eukaryotic systems, it is currently being pursued as a target for a novel anti- bacterial strategy. Very recently, biochemical studies have established that PDF represents an intriguing new class of amide hydrolyase, which utilizes a ferrous ion as the catalytic metal (7–9). The extraordinary lability of PDF, which prevented its purification or characterization for three decades, was shown to be due to sensitivity of the catalytic Fe 2+ ion to environ- mental oxygen (10). Through in vitro reconstitution or overexpression in defined media, the iron metal has been replaced by zinc (7, 8, 11), nickel (9, 12), or cobalt (P.T.R.R. and D.P., unpublished results) to give stable variants that retain partial (Zn form) or nearly full catalytic activity (Ni and Co forms). The availability of these stable enzyme forms has permitted the struc- tural characterization of this enzyme. The data from several laboratories reveal that the Zn 2+ , Ni 2+ , Co 2+ , and more recently the Fe 2+ forms are virtually identi- cal in three-dimensional structure (13–18). The metal ion is tetrahedrally bound by a water molecule and the side chains of two histidines of the conserved HEXXH motif and of a conserved cysteine. Catalysis appears to be mediated by the direct attack on the substrate car- bonyl by the metal-bound water, which is ionized upon substrate binding (17, 18). To facilitate the mechanistic study of PDF and in- hibitor screening, we (8, 19) as well as others (20) have previously reported two different assay methods for this enzyme. One method monitors the release of for- mate, using a formate dehydrogenase as the coupling enzyme. The drawback of this assay is that, because of the poor specific activity of the dehydrogenases, a large amount of the dehydrogenase must be used in each assay, making it financially undesirable for screening a large number of inhibitors (8, 20). The second method 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. Fax: (614) 292- 1532. E-mail: pei.3@osu.edu. 3 Abbreviations used: PDF, peptide deformylase; DTNB, 5,5'-di- thio-bis(2-nitrobenzoic acid); f-ML-pNA, N-formylmethionylleucyl-p- nitroanilide; Fe 2+ -PDF, Fe 2+ -containing deformylase; DABCO, diazabicyclo[2,2,2]octane. 298 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 273, 298 –304 (1999) Article ID abio.1999.4239, available online at http://www.idealibrary.com on