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 NH2terminus, a substrate for an aminopeptidase fromAeromonas proteolytica.Sequential hydrolysis of the dipeptide by the aminopeptidase releases ap-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 fromEscherichia coli.TheE. colienzyme is most active near neutral pH (pH 7.0) and displays Michaelis–Menten kinetics toward the formylated dipeptide, withKM= 20.3 ± 1.3 μm,kcat= 38 ± 2 s−1, andkcat/KM= 1.9 × 106m−1s−1. It also exhibits an acylase activity, capable of deacylatingN-acetyl-Met-Leu-p-nitroanilide andN-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.