A continuous fluorimetric assay for aminopeptidase P detailed analysis of product inhibition

Abstract

Aminopeptidase P (APP; EC 3.4.11.9) is a proline-specific peptidase hydrolyzing N-terminal Xaa-Pro peptide bonds. On the basis of its unique substrate specificity it is difficult to determine enzyme activity and to estimate potential enzyme inhibitors. In this report, we describe the synthesis of a new fluorogenic substrate to assay APP. The substrate was characterized using Escherichia coli APP and rat intestine APP. The compound contains the fluorogenic 2-aminobenzoyl residue and 4-nitroanilide as internal quencher. Both enzymes hydrolyze the substrate Lys( N ε -2-aminobenzoyl)-Pro-Pro-4-nitroanilide at the Lys-Pro peptide bond with K m values in the micromolar range. Lys( N ε -2-aminobenzoyl)-Pro-Pro-4-nitroanilide is the best substrate of APP from rat intestine that is known with a K m value of 3.54 μM and a second-order rate constant of 1,142,000 M −1 s −1. Unfortunately, product inhibition occurs. Inhibition studies using the hydrolysis product Pro-Pro-4-nitroanilide demonstrate a linear mixed-type mechanism with a K i value of 20.8 μM and an α value of 5.1 for rat APP and a K i value of 76.1 μM and an α value of 0.4 for E. coli APP, respectively. Furthermore, the hydrolysis of the fluorogenic APP substrate catalyzed by prolyl oligopeptidase was investigated.