Details

Autor(en) / Beteiligte

• Campbell, Benedict J.
• Yuan, Di Shih
• Forrester, Lawrence J.
• Zahler, Warren L.

Titel

Specificity and inhibition studies of human renal dipeptidase

Ist Teil von

• Biochimica et biophysica acta, 1988-09, Vol.956 (2), p.110-118

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

1988

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and trippetidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-colomn derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of V max/ K m for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accomodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-tate peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were obseved. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the β-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leuktriene D 4 to leukotriene E 4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D 4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay. The specific activity of the enzyme at 0.1 mM leukotriene D 4 was 12.1 μmol leukotriene D 4 per min per mg enzyme.