title: Continuous spectrophotometric assay of peptide deformylase.
abstract: A continuous assay for peptide deformylase has been developed using a formylated dipeptide, formyl-Met-Leu-p-nitroanilide, as substrate. Removal of the formyl group by a peptide deformylase renders the dipeptide product, which contains a free NH2 terminus, a substrate for an aminopeptidase from Aeromonas proteolytica. Sequential hydrolysis of the dipeptide by the aminopeptidase releases a p-nitroaniline, which is monitored spectrophotometrically at 405 nm. This assay was applied to determine the pH optimum and the catalytic activity of a peptide deformylase from Escherichia coli. The E. coli enzyme is most active near neutral pH (pH 7.0) and displays Michaelis-Menten kinetics toward the formylated dipeptide, with K(M) = 20.3 +/- 1.3 microM, k(cat) = 38 +/- 2 s(-1), and k(cat)/K(M) = 1.9 x 10(6) M(-1) s(-1). It also exhibits an acylase activity, capable of deacylating N-acetyl-Met-Leu-p-nitroanilide and N-trifluoroacetyl-Met-Leu-p-nitroanilide, albeit at drastically reduced rates. These results demonstrate that the current assay is a convenient, rapid, and sensitive method for kinetic studies of peptide deformylase. The strategy employed in this work should also be generally applicable to the characterization of other acylases.
authors.affiliation: Ohio State Biochemistry Program, The Ohio State University, Columbus 43210, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Wei
authors.forename: D
authors.initials: D
authors.lastname: Pei
mesh_terms: D000333:Aeromonas / Q000201:enzymology; D000581:Amidohydrolases; D000626:Aminopeptidases / Q000378:metabolism*; D000814:Aniline Compounds / Q000032:analysis; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D000814:Aniline Compounds; D004151:Dipeptides; C019498:4-nitroaniline; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase