L-Proline 4-nitroanilide

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.

Six aminopeptidases differing in enzymic specificity against various L-amino acid-4-nitroanilides were detected and isolated from the cytosol of leucocytes collected from the buffy coat of human blood. The different enzymes were separated by one step of chromatography on DEAE-Sephacel and were further purified by gel filtration on Sephacryl S-300. The main aminopeptidases of the cytosol were designated aminopeptidases 1, 2, 4 and 5 (AP 1, AP 2, AP 4, AP 5) on the basis of their elution sequence from the first ion-exchange chromatography column on DEAE-Sephacel. Aminopeptidase 1 appeared to be a strongly sulfhydryl-dependent leucine aminopeptidase, activatable by thiol reagents. The enzyme was inhibited by p-chloromercuribenzoate. Its molecular mass was estimated to be 150 kDa. Aminopeptidase 2 showed high specificity for proline-4-nitroanilide. This enzyme was inhibited by p-chloro-mercuribenzoate and bestatin. It exhibited a molecular mass of 70 kDa. Aminopeptidase 4 designated the activities of two different enzymes of apparent molecular masses of 220 and 70 kDa which could be further separated by gel filtration. Aminopeptidase 5 exhibited the properties alike aminopeptidase B with high specific hydrolytic activity against the 4-nitroanilides of lysine and arginine. The molecular mass was estimated to be 90 kDa. Aminopeptidase 3 was a minor component in the cytosol and could be identified as an extracellular leucocyte plasma membrane constituent. The enzyme exhibited properties of a metallo proteinase and could be inhibited by EDTA. The molecular mass was estimated to be 250 kDa.

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.