No abstract

Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp 641 residue of the catalytic triad (Ser 554 , Asp 641 , His 680 ) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k cat /K m for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k cat /K m . This indicates that the contribution of Asp 641 is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His 680 with Z-Gly-Pro-CH 2 Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His 680 is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.

Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2 region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1 amide and P2 carbonyl groups and another between the P2 amide and N⑀2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the N⑀2 of His 680 . Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK a of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pK a for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k cat /K m , and indicated that the observed pK a values are apparent. The procedure presented may be applicable for other serine peptidases.Prolyl oligopeptidase (EC 3.4.21.26), previously called prolyl endopeptidase or post-proline cleaving enzyme, is a large intracellular enzyme (molecular mass 80 kDa) that preferentially hydrolyzes proline-containing peptides at the carboxyl end of proline residues (1-3). It is presumably involved in the maturation and degradation of peptide hormones and neuropeptides (1). Prolyl oligopeptidase has recently gained pharmaceutical interest, because specific inhibitors reverse scopolamine-induced amnesia in rats (4 -6). Its activity in plasma correlates with different stages of depression (7). The enzyme also has a role in the regulation of blood pressure by participating in the renin-angiotensin system through metabolism of bradykinin and angiotensin I and II (8).Prolyl oligopeptidase is unrelated to the well known trypsin and subtilisin families and belongs to a new class of serine peptidases (clan SC, family S9), which also includes dipeptidyl peptidase IV, acylaminoacyl peptidase, and oligopeptidase B (9, 10). These enzymes display distinct specificities, and each contains a peptidase domain at the carboxyl-terminal region of the single polypeptide chain. In the case of prolyl oligopeptidase, the active site serine and histidine have been identified as Ser 554 and His 680 , respectively (11, 12). A structural relationship between lipases and the peptidase domain of oligopeptidases has been indicated by the similar topology of the catalytic groups and by the homologous amino acid sequences around these residues (13). The mechanism of action of serine peptidases involves an acyl enzyme intermediate. Both the formation and the decomposition of the acyl enzyme proceed through the formation of a negatively charged tetrahedral interm...

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.