Continuous spectrophotometric assay of peptide deformylase.

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.