RESUMO
Novel, potent inhibitors of aminopeptidase P, containing a 3-amino-2-hydroxy acid and a proline or a proline analogues, have been prepared. One part of the bestatin-derived inhibitors was found to inhibit APP from Escherichia coli and from rat intestine according to a mixed-type mechanism, with Ki values up to 1.26 microM. The other compounds, 3-amino-2-hydroxy acyl prolines of a different configuration, inhibit APP competitively, according to a slow-binding mechanism, with Ki values in the nanomolar up to the micromolar range.
Assuntos
Aminoácidos/química , Aminoácidos/farmacologia , Aminopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Animais , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Cinética , Leucina/análogos & derivados , Leucina/química , Leucina/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , Pirróis/química , Pirróis/farmacologia , Ratos , Relação Estrutura-Atividade , Especificidade por Substrato , Tiazóis/química , Tiazóis/farmacologiaRESUMO
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.
