A continuous fluorimetric assay for aminopeptidase P detailed analysis of product inhibition.
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(epsilon)-2-aminobenzoyl)-Pro-Pro-4-nitroanilide at the Lys-Pro peptide bond with Km values in the micromolar range. Lys(N(epsilon)-2-aminobenzoyl)-Pro-Pro-4-nitroanilide is the best substrate of APP from rat intestine that is known with a Km value of 3.54 microM 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 microM and an alpha value of 5.1 for rat APP and a K(i) value of 76.1 microM and an alpha value of 0.4 for E. coli APP, respectively. Furthermore, the hydrolysis of the fluorogenic APP substrate catalyzed by prolyl oligopeptidase was investigated.
Stöckel-Maschek
Angela
A
Department of Biochemistry and Biotechnology, Institute of Biochemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle/Saale, Germany. stoeckel@biochemtech.uni-halle.de
Stiebitz
Beate
B
Koelsch
Regine
R
Neubert
Klaus
K
D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D007422:Intestines / Q000201:enzymology; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry; D000085822:Prolyl Oligopeptidases; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D010455:Peptides; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases