Structures of Prolyl Oligopeptidase Substrate/Inhibitor Complexes

Abstract

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...

Electrostatic Environment at the Active Site of Prolyl Oligopeptidase Is Highly Influential during Substrate Binding

“…Nevertheless, during the long crystallization period the substrates were cleaved so that only the acyl parts of the substrates were seen in the electron density maps. These enzyme-product complexes share identical binding modes to the enzyme-substrate complexes determined previously (18). In the case of subtilisin modified also at the active site serine residue, an ϳ10 6 -fold decrease in activity was demonstrated with kinetic analysis (34), indicating a small remaining activity, but with a different mechanism.…”

“…Therefore, we examined the effects on the kinetic and thermodynamic parameters of the glutamic acid residue located at various sites of the leader peptide, Abz-Gly-Phe-Ser-ProPhe(NO 2 )-Arg-Ala, which corresponds to the P1-P4 amino acids of bradykinin, a possible natural substrate. Our previous x-ray crystallographic studies on the binding of an octapeptide indicated that the P3-P2Ј residues were clearly associated with the enzyme, whereas the P4 residue might bind weakly (18). Yet, we have also extended the position of Glu toward subsites 4 and 5, because it is known from the three-dimensional structure of prolyl oligopeptidase (20) that Arg-252 and Arg-128 are not far from the binding site so that they may form appropriate subsites for the binding of Glu in those positions.…”

“…The crystal structures assumed similar binding modes for all substrates involved in important interactions with the peptide bonds of the P2 and P3 residues, as described earlier (16,18). Of course, the P1 Pro ring interacting with the side chain of Trp-595 determines the primary specificity.…”

“…5). Instead of the wild type enzyme, we used the inactive prolyl oligopeptidase in which an alanine residue replaced the catalytic serine (18). Nevertheless, during the long crystallization period the substrates were cleaved so that only the acyl parts of the substrates were seen in the electron density maps.…”

“…The complex electrostatic environment created by these residues may considerably influence the binding and thus the specificity of substrates. We have determined previously that, principally, five subsites of the enzyme (S3-S2Ј) interact with a polypeptide substrate (18). In this work we have examined the effects of charged residues at different subsites.…”