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

Abstract

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pK a values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of k cat /K m did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k 1 ) and decomposition (k ؊1 ) of the enzyme-substrate complex and the acylation constant (k 2 ), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k 1 rather than k 2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.Prolyl oligopeptidase is a member of a relatively new group of serine peptidases unrelated to the well known trypsin and subtilisin families (1-4). The new family includes enzymes of different specificities like the prolyl oligopeptidase itself, dipeptidyl-peptidase IV, acylaminoacyl-peptidase, and oligopeptidase B (5). These enzymes selectively cleave substrates that are no longer than ϳ30 amino acid residues in total. Prolyl oligopeptidase (EC 3.4.21.26) is implicated in the metabolism of peptide hormones and neuropeptides (6 -8). Because specific inhibitors relieve scopolamine-induced amnesia (9 -12), the enzyme is of pharmaceutical interest. The activity of prolyl oligopeptidase has also been associated with depression (13,14) and blood pressure regulation (15).The crystal structure determination of prolyl oligopeptidase has revealed that the carboxyl-terminal peptidase domain of the enzyme displays an ␣/␤ hydrolase fold and that its catalytic triad (Ser-554, His-680, and Asp-641) is covered by the central tunnel of an unusual ␤-propeller (16). Recent engineering of the enzyme provided evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins, thereby preventing accidental proteolysis in the cytosol (17).The active site region of prolyl oligopeptidase exhibits several charged residues such as Arg-643, Asp-642, Arg-252, Asp-149, and Arg-128. The complex electrostatic environment created by these residues may considerably influence the binding...

The Acylaminoacyl Peptidase from Aeropyrum pernix K1 Thought to Be an Exopeptidase Displays Endopeptidase Activity

“…In the case of the related prolyl oligopeptidase, the electrostatic environment of the active site region also considerably affected the pHrate profiles determined with various charged substrates. 13 ApAAP displays both exo-and endopeptidase activities, as well as sequence-dependent S1 specificity…”

“…A similar approach was employed successfully for prolyl oligopeptidase, 23 but in another study the "inactive" enzyme hydrolysed the substrate. 13 Likewise, the substrate was cleaved during the lengthy crystallization period in the present investigation, so that only its acyl portion was seen in the crystal structure ( Figure 8). In the case of subtilisin modified similarly at the catalytic serine residue, an ∼10 6 -fold decrease in activity was demonstrated, indicating a small remaining activity, but with different mechanism not involving an acyl-enzyme intermediate.…”

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

“…3 and Table1) mutations in the flexible loop structure markedly changed the catalytic power of POP resulting in orders of magnitude differences in activity. The pH-optimum of POP was also changed considerably, suggesting that mutations alter the catalytically relevant electrostatic interactions at the active site, which can markedly affect the binding and the route of the substrates to active site of POP [42]. A finely regulated functioning of the loop structure seems to be required for the mildly alkaline pH optimum of POP, at least with the neutral substrates like the short Z-Gly-Pro-BNA and the octapeptide.…”

Discrimination of Esterase and Peptidase Activities of Acylaminoacyl Peptidase from Hyperthermophilic Aeropyrum pernix K1 by a Single Mutation

“…Crystal structures of prolyl oligopeptidase-substrate complexes from porcine muscle reveal that Arg involves itself in substrate binding by forming a hydrogen bond between the guanidine group and the main chain carbonyl group of the substrate (13). Arg 526 pulls the negatively charged substrate away from the catalytically competent position (4). Moreover, structure-based mutagenesis of the Myxococcus xanthus prolyl oligopeptidase has revealed that Arg 526 is involved in a salt bridge that acts as a latch for opening or closing the enzyme, and Arg 526 also helps to secure the incoming peptide…”