Purification and characterization of the sunflower seed (Helianthus annuus L.) major aminopeptidase

Abstract

The sunflower seed (Helianthus annuus L.) major peptidase was purified to molecular homogeneity. It is an 80 kDa enzyme with pI of 4.6 and optimal activity at pH 7.5-8.0 and 45-50°C. It is a thiol-dependent aminopeptidase hydrolyzing peptides in a step-by-step manner as cleaving after the N-terminal amino acid residue of the substrate. It requires substrate acyl parts with a free amino group in either a-or b-position and L-configuration of the adjacent carbon atom. The enzyme prefers amino acid residues with bulky hydrophobic side chains at P 1 -position and its catalytic efficacy is affected by the structure of both P 1 and P 1 0 parts of the substrate.

Purification, biochemical characterisation, and mass spectrometry analysis of phenylalanine aminopeptidase from the shoots of pea plants

“…In contrast, AP1 from pea seeds was not suppressed in the presence of serine peptidase inhibitors (Elleman 1974). The metallopeptidase inhibitors EDTA and 1,10-phenanthroline were found to be ineffective at reducing the activity of Phe-AP and aromatic aminopeptidases isolated from wheat leaves, grapes, sunflower seeds, and cotyledons of cowpea (Kang et al 1999;Tishinov et al 2009;Waters and Dalling 1984;Wynn and Murray 1985). This disproves the idea that aromatic aminopeptidases belong to the group of leucine aminopeptidases, which are classified as belonging to the M17 metallopeptidase family (Matsui et al 2006;Walling and Gu 1996).…”

“…This enzyme belongs to the family of aminopeptidases that preferentially hydrolyse the substrates with N-terminal aromatic amino acids. Despite a number of publications that confirm the existence of this group of enzymes, no one has so far undertaken to define aromatic aminopeptidases as a separate group (Elleman 1974;Kang et al 1999;Kolehmainen and Mikola 1971;Ogiwara et al 2005;Tishinov et al 2009;Vodkin and Scandalios 1980;Waters and Dalling 1984). Instead, based on their ability to hydrolyse Leu-b-naphthylamide and Leu-p-nitroanilide substrates, these enzymes are often categorised as leucine aminopeptidases (EC 3.4.11.1;Collier and Murray 1977; The enzyme was pre-incubated in 50 mM Tris-HCl buffer (pH 7.5) containing different inhibitors for 60 min at room temperature; Phe-b-NA was then added and the enzymatic reaction was performed at 37°C Ogiwara et al 2005).…”

“…The Phe-AP from pea shoots was purified to homogeneity 513-fold. It needs to be emphasised that only in some studies was a homogenous enzyme investigated (Elleman 1974;Tishinov et al 2009); in the majority of cases enzyme activity was observed and characterised in crude, partially purified, or considerably purified extracts (Collier and Murray 1977;Kolehmainen and Mikola 1971;Ogiwara et al 2005;Wynn and Murray 1985). Unfortunately, due to the low stability of the enzyme, each stage of purification caused a drop in the total activity, and the final recovery was 8%.…”

“…(Elleman 1974;Kang et al 1999;Kolehmainen and Mikola 1971;Tishinov et al 2009;Waters and Dalling 1984). The majority of aromatic aminopeptidases are partially suppressed in the presence of serine peptidase inhibitors, such as PMSF and/or DFP, which suggests the participation of the hydroxyl groups in catalysis or in the maintenance of protein stability (Kang et al 1999;Ogiwara et al 2005;Tishinov et al 2009). The impact of the applied diagnostic inhibitors of serine and thiol-dependent proteases on Phe-AP from pea shoots confirms that it belongs to the group of aromatic aminopeptidases; however, the effect of the factors that modify the hydroxyl groups is much stronger in comparison to other enzymes from this group.…”

“…Aromatic aminopeptidases are characterised by low K m values against substrates with Phe, Trp, or Tyr (but mainly Phe) at the N-terminus, a considerable but significantly lower activity against Leu or Met, and little or undetectable activity against Gly, Ala, Glu, Arg, or Lys (Kang et al 1999;Kolehmainen and Mikola 1971;Tishinov et al 2009;Vodkin and Scandalios 1980;Waters and Dalling 1984;Wynn and Murray 1985;Yamaoka et al 1994). Most of these enzymes are also partially inhibited by serine protease inhibitors, and their activities remain unchanged in the presence of metal chelators (Kang et al 1999;Ogiwara et al 2005;Tishinov et al 2009;Yamaoka et al 1994). There is no sequence (amino acid or nucleotide) or crystallographic data available for any of the members of this thiol-dependent group of plant aminopeptidases, which considerably hinders the establishment of their phylogenetic relations with other proteins and their proper classification.…”