Assay for rapid analysis of the tri-peptidase activity of LTA4 hydrolase

Leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A(4) hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tripeptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A(4) hydrolase as well as other members of the M1 family of aminopeptidases. Proteins 2007;67:1113-1118. (C) 2007 Wiley-Liss, Inc.