Abstract
Aspergilloglutamic peptidase (formerly called aspergillopepsin II) is an acid endopeptidase produced by Aspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, thus belonging to a novel peptidase family G1. The mature enzyme is generated from its precursor by removal of the putative 41-residue propeptide and an 11-residue intervening peptide through autocatalytic activation. In the present study, the propeptide (Ala1-Asn41) and a series of its truncated peptides were chemically synthesized, and their effects on the enzyme activity and thermal stability were examined to identify the sequences and residues in the propeptide most critical to the inhibition and thermal stabilization. The synthetic propeptide was shown to be a potent competitive inhibitor of the enzyme (Ki = 27 nM at pH 4.0). Various shorter propeptide fragments derived from the central region of the propeptide had significant inhibitory effect, whereas their Ala scan-substituted peptides, especially R19A and H20A, showed only weak inhibition. Substitution of the Pro23-Pro24 sequence near His20 with an Ala-Ala sequence changed the peptide Lys18-Tyr25 to a substrate with His20 as the P1 residue. Furthermore, the propeptide was shown to be able to significantly protect the enzyme from thermal denaturation (DeltaTm = approximately 19 degrees C at pH 5.6). The protective potencies of the propeptide as well as truncated propeptides and their Ala scan-substituted peptides are parallel with their inhibitory potencies. These results indicate that the central part, and especially Arg19 and His20 therein, of the propeptide is most critical to the inhibition and thermal stabilization and that His20 interacts with the enzyme at or near the S1 site in a nonproductive fashion.
Highlights
Aspergilloglutamic peptidase is an acid endopeptidase produced by Aspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, belonging to a novel peptidase family G1
These results indicate that the central part, and especially Arg[19] and His[20] therein, of the propeptide is most critical to the inhibition and thermal stabilization and that His[20] interacts with the enzyme at or near the S1 site in a nonproductive fashion
The results indicate that the propeptide inhibits the enzyme competitively with a Ki value of 0.027 M
Summary
The protective potencies of the propeptide as well as truncated propeptides and their Ala scan-substituted peptides are parallel with their inhibitory potencies These results indicate that the central part, and especially Arg[19] and His[20] therein, of the propeptide is most critical to the inhibition and thermal stabilization and that His[20] interacts with the enzyme at or near the S1 site in a nonproductive fashion. Comparison of the effect of inhibition with that of thermal stabilization by a series of the truncated propeptides revealed that the central part (Lys18– Tyr25), especially Arg[19] and His[20] therein, is most important for both these effects This is the first time that a short peptide segment and some residues therein of the propeptide of a. Propeptide Inhibition/Stabilization of AGP peptidase were shown to be critical to both enzyme inhibition and stability
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