Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P₂, P₁ and P₂′ positions

Abstract

The yeast aspartic protease Yap3 is localised to the secretory pathway and correctly cleaves pro-a-mating factor at its dibasic sites. We determined the specificity of Yap3 for mono-, di-and multi-basic cleavage sites in the context of 15 residue synthetic proalbumin peptides. Yap3 cleaved after dibasic ArgArg and LysArg sites but not after monobasic Arg sites even when there was an additional arginine at -6 and/or -4. Yap3 did not cleave a tetra-arginine site and tribasic sites (RRR and RRK) were poor substrates. Cleavage always occurred C-terminal to the last arginine in the di-or tri-basic sequence. The optimal cleavage site sequence was RR ~DR and this substrate was cleaved 8-9-fold faster than the normal RR $ DA sequence. In contrast to Kex2, Yap3 did not remove the propeptide from normal proalbumin or a range of natural or recombinant proalbumin variants. However at pH 4.0 Yap3 slowly cleaved proalbumin and albumin between domains 2 and 3.

Synthesis and Characterization of the First Potent Inhibitor of Yapsin 1

“…Extensive analyses of its specificity (15,(22)(23)(24) have revealed that substrates containing multiple basic residues surrounding the scissile bond are cleaved with high efficiencies. The nature of this specificity is not completely understood even though molecular modeling (Protein Data Bank code 1YPS) (24) has revealed that yapsin 1 has a more open active site compared with other aspartyl proteases and that many of its subsites are electronegative, thus allowing for the accommodation of positively charged residues within and surrounding the cleavage site (24).…”

Activation and Processing of Non-anchored Yapsin 1 (Yap3p)

“…In the same expression system, yapsin 1 activity was also found in the culture supernatant and was characterized as ϳ150 -180-and ϳ90-kDa hyperglycosylated forms that appeared to be highly stable (8). Ϫ1 for human adrenocorticotropin (ACTH ) (15), demonstrating an enhancement of the efficiency by additional basic residues upstream and especially downstream of the cleavage site (15,16).…”

“…The intermediate form undergoes a further intermolecular or intramolecular cleavage of the remainder of the proregion to generate the mature active enzyme. It is interesting to note the similarity to yapsin 1 in this respect in that a potential cleavage site exists at Lys 16 -Phe 17 within the propeptide. Because of this structural similarity and that cleavage of a mono-lysine residue by yapsin 1 has previously been documented (15), it is predicted that a yapsin 1 intermediate, formed from an intramolecular cleavage presumably at this site, exists.…”

“…For pepsin, the model aspartic protease, its proregion, which contains 11 basic residues, is partially stabilized by ionic interactions with the negatively charged aspartic acid residues of the active site (41). Upon acidification, the aspartic acid residues become protonated and less charged (pK a ϭ 4.3) causing destabilization and an intramolecular cleavage at Leu 16 -Ile 17 within the propeptide resulting in the generation of an intermediate form of the enzyme or pseudo-pepsin. The intermediate form undergoes a further intermolecular or intramolecular cleavage of the remainder of the proregion to generate the mature active enzyme.…”

“…B, from the studies of the baculovirus-expressed proyapsin 1, an intermediate or pseudo-yapsin 1 has been identified presumably as a result of a selfcleavage at Lys 37 (i.e. Lys 16 of the propeptide). C, the remainder of the proregion is then likely to be removed at the Lys 66 -Arg 67 cleavage site by an intra-molecular mechanism.…”