The Noncatalytic β-Propeller Domain of Prolyl Oligopeptidase Enhances the Catalytic Capability of the Peptidase Domain

Abstract

Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a beta-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 --> Thr, Cys-255 --> Ala, and Cys-255 --> Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 --> Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly-Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO(2))-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl-Gly-Pro-OH and aminobenzoyl-Ser-d-Pro-Phe(NO(2))-Ala-OH. It was concluded that, besides selecting substrates by size, the beta-propeller domain containing Cys-255 remarkably contributed to catalysis of the peptidase domain.

Structures of Prolyl Oligopeptidase Substrate/Inhibitor Complexes

“…Unlike the sigmoid or bell-shaped pH rate profiles observed with the classic serine peptidases, the pH dependence for prolyl oligopeptidase is more complicated. This has previously been shown with Z-Gly-Pro-Nap (24,25) and also demonstrated here with the octapeptide substrate. As seen in Fig.…”

The prolyl oligopeptidase family

“…Unlike the sigmoid or bell-shaped pHrate profiles observed with the classic serine peptidases, the pH dependence for prolyl oligopeptidase is more complicated. This has been shown with several substrates [60,62,63]. As seen in figure 3A, the data conform to a doubly bell shaped curve, which arises from modification of the usual bell-shaped curve by an additional ionization event involving a group with an apparent pK a of ~7.…”

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

“…In contrast, the loop A (T202C) and loop B (T590C) point mutants displayed altered ligand binding but relatively unaltered catalytic activity. Different again, in previous studies the Asp loop (D641A) and the Cys loop (C255A) variants showed altered pH dependencies, but ligand binding was only slightly affected [40,43]. This implies a much greater corruption of the active site structure in the loop A -variant.…”

“…The further change in the preference for ZGly-Pro-BNA over the longer octapeptide upon air and glutathione oxidation is presented in Table 2. The oxidized glutathione partially inactivated (30%) the wild-type POP too, but equally for both the shorter and the longer peptide, while the C255S control variant not containing the C255 responsible for the thiol-sensitivity of POP activity [40] retained the initial activity. 89 % and 87% of the activity of the glutathione-oxidized and air-oxidized T202C/T590C variant could be recovered with a DTT treatment (10 mM), respectively.…”