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. Rem oval of the formyl group by a peptide deformylase renders the dipeptid e product, which contains a free NH2 terminus, a substrate for an amin opeptidase from Aeromonas proteolytica. Sequential hydrolysis of the d ipeptide by the aminopeptidase releases a p-nitroaniline, which is mon itored spectrophotometrically at 405 nm. This assay was applied to det ermine the pH optimum and the catalytic activity of a peptide deformyl ase from Escherichia coli. The E. coli enzyme is most active near neut ral pH (pH 7.0) and displays Michaelis-Menten kinetics toward the form ylated dipeptide, with K-M = 20.3 +/- 1.3 mu M, 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 reduce d rates. These results demonstrate that the current assay is a conveni ent, rapid, and sensitive method for kinetic studies of peptide deform ylase. The strategy employed in this work should also be generally app licable to the characterization of other acylases. (C) 1997 Academic P ress.