Biochemical characterisation of prolyl aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions

Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54 kDa. Its pH and temperature optimum are pH 7.5 and 37 °C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β-naphthylamides (β-NA) of Ala, Phe, and Leu. The Km value of PAP against Pro-β-NA was the lowest among the substrates tested and it was 1.47 × 10−5 M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-β-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.

► A multimeric prolyl aminopeptidase (PAP) was purified from triticale shoots.
► The enzyme highly prefers substrates with Pro and Hyp at the N-terminus.
► The most effective inhibitors of PAP activity are the serine protease inhibitors.
► The aminopeptidase activity against Pro-β-NA increases in response to stress conditions.