Novel Inhibitor for Prolyl Tripeptidyl Aminopeptidase from Porphyromonas gingivalis and Details of Substrate-recognition Mechanism

Abstract

Prolyl Tripeptidyl Aminopeptidase from P. gingivalis 2 SummaryA new inhibitor, H-Ala-Ile-pyrrolidin-2-yl boronic acid, was developed as an inhibitor against prolyl tripeptidyl aminopeptidase with the K i value of 88.1 nM. The structure of the prolyl tripeptidyl aminopeptidase complexed with the inhibitor (enzyme-inhibitor complex) was determined at 2.2 Å resolution. The inhibitor was bound to the active site through a covalent bond between Ser603 and the boron atom of the inhibitor. This structure should closely mimic the structure of the reaction-intermediate between the enzyme and substrate.We previously proposed that two glutamate residues, Glu205 and Glu636, are involved in the recognition of substrates. In order to clarify the function of these glutamate residues in substrate recognition, three mutant enzymes, E205A, E205Q, and E636A were generated by site-directed mutagenesis. The E205A mutant was expressed as an inclusion body. The E205Q mutant was expressed in soluble form, but no activity was detected for it.Here, the structures of the E636A mutant and its complex with the inhibitor were determined. The inhibitor was located at almost the same position as in the wild-type enzyme-inhibitor complex. The amino group of the inhibitor interacted with Glu205 and the main-chain carbonyl group of Gln203. In addition, a water molecule in the place of Glu636 of the wild-type enzyme interacted with the amino group of the inhibitor. This water molecule was located near the position of Glu636 in the wild type and formed a hydrogen bond with Gln203. The k cat /K M values of the E636A mutant toward the two substrates used were smaller than those of the wild type by two Prolyl Tripeptidyl Aminopeptidase from P. gingivalis 3 orders of magnitude. The K i value of our inhibitor for the E636A mutant was 48.8 M, which was 554-fold higher than that against the wild-type enzyme. Consequently, it was concluded that Glu205 and Glu636 are significant residues for the N-terminal recognition of a substrate.

A Monosaccharide‐Based Coin‐Cell Biobattery

“…However, distances of 3.8 Å ( T. brucei TR) and 4.0 Å ( T. cruzi TR) between the oppositely charged atoms indicate a long‐range Coulombic interaction rather than a strong charge‐assisted hydrogen bond. This correlates with the observation of an only 3.5‐fold increased K ic value of the Glu 18 Ala mutant relative to the wild‐type enzyme 53. In both TR species, Glu 467′ is not involved in binding.…”

Hybrid molecular structure of the giant protease tripeptidyl peptidase II

“…These two glutamates resemble the double–Glu motifs found in Dipeptidyl peptidase IV22, Tricorn factor F123 and Prolyl–tripeptidylpeptidase24,25 which coordinate the positively charged N–terminus of peptide substrates via ionic interactions. Therefore, we mutated either of the two glutamates into glutamine residues to probe their potential interaction with the substrate and found that both substitutions lead to a drastic increase in K M and decrease of the cleavage rate (Supplementary Table 1).…”

“…This is a recurrent structural principle among aminopeptidases and the two glutamate residues appear to form the double–Glu motif that has emerged as the prevailing motif for imparting aminopeptidase activity on peptidases, such as Dipeptidylpeptidase IV22, Aminopeptidase N32, and Aminopeptidase F123. Among tripeptidyl peptidases, this motif has recently been described in prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis24,25. Based on homology to this peptidase, the double–Glu motif had been suggested to be present in TPP II33.…”

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

“…Our models suggest that SpAAP is deprived of these covalent interactions, pointing out another structural determinant of the cold adaptation of SpAAP. A lack of disulphide bridges in favour of free cysteines is also observed in the structures of mesophilic AAPs, such as Streptomyces morookaense (PDB entry 3AZO [43]) and Phorphyromonas gingivalis AAPs (PDB entry 2Z3Z [53]). …”