engleski

Two Intracellular Proline-specific Aminopeptidases from Streptomyces rimosus

In mycelium and culture filtrate of the bacterium Streptomyces rimosus, the industrial producer of antibiotic oxytetracycline, a hydrolytic activity towards proline-2-naphthylamide (Pro-2NA) was detected. During extraction and separation of proteins from the mycelium, the presence of two aminopeptidases responsible for this activity was revealed. The one eluted first from ion exchange column (AP I) was partially purified, and the second one (AP II) was purified to homogeneity, and their properties were examined. Both enzymes hydrolyzed exclusively arylamides, dipeptides and tripeptides having at N-terminus L-proline, and with lower efficiency those having hydroxyproline, which classifies them as real prolyl aminopeptidases (Pro-AP). However, Pro-AP I was less permissive for hydroxyproline than Pro-AP II. Dipeptides having glutamic acid or proline at the second position were not hydrolyzed. These two enzymes also differed by Km for Pro-2NA and proline-p-nitroanilide, by pH, temperature and ionic strength optimal for the catalytic reaction, and by pH and temperature stability. Pro-AP I was less stable, had lower affinity for proline-p-nitroanilide and did not tolerate salt concentrations above 0.2 M, that were required for the maximal activity of Pro-AP II. The most pronounced difference between the two enzymes was observed in their susceptibility to various peptidase inhibitors. Activity of Pro-AP I was abolished by 0.5 mM o-phenanthroline, while Pro-AP II retained its full activity. On the other hand, 1 mM 3, 4-dichloroisocoumarin inhibited more efficiently Pro-AP II than Pro-AP I. The both enzymes responded to the presence of 1 mM iodacetamide, Pro-AP I being more sensitive to that inhibitor. Based on these results Pro-AP I would be metallopeptidase requiring free SH-group for its activity, and Pro-AP II most probably serine peptidase with cysteine in its active center.

Streptomyces rimosus; Prolyl aminopeptidase

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