Low Barrier Hydrogen Bond Is Absent in the Catalytic Triads in the Ground State but Is Present in a Transition-state Complex in the Prolyl Oligopeptidase Family of Serine Proteases

Kahyaoglu, Ara; Haghjoo, Khadijeh; Guo, Feng; Jordan, Frank; Kettner, Charles A.; Felföldi, Ferenc; Polgár, László

doi:10.1074/jbc.272.41.25547

Cited by 29 publications

(31 citation statements)

References 36 publications

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“…We found the same resonance in all of our mutant proteins, free and complexed with either inhibitor, suggesting that this signal does not arise from an active-site residue. A similar observation has been reported for serine proteases belonging to the class of prolyl oligopeptidases: prolyl oligopeptidase (PO) and oligopeptidase B of Escherichia coli (OpB) (Kahayaoglu et al 1997). Downfield chemical shifts (;17 and ;16 ppm) were observed for the wild type and an active-site H652A mutant of OpB.…”

Section: Y547fsupporting

confidence: 55%

Involvement of DPP‐IV catalytic residues in enzyme–saxagliptin complex formation

et al. 2008

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The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 Å ). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an ;1000-fold reduction in affinity relative to DPP-IV WT , while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at ;14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.Keywords: DPP-IV; X-ray crystal structure; mutant; ITC; proton NMR; short, strong hydrogen bond; serine protease; saxagliptin Dipeptidyl peptidase IV is a serine protease that modulates the biological activity of specific circulating peptide hormones, chemokines, cytokines, and neuropeptides Abbreviations: ANS, 1-anilino-8-naphthalene sulfonate; DPP-IV, dipeptidyl peptidase IV; GLP-1, glucagon-like peptide-1; ITC, isothermal titration calorimetry; pNA, p-nitroaniline; P n -P n9 , amino acid residues of the substrate which numerically indicate the position relatively to the scissile bond, n being the residues toward the N terminus and n9 being the residues toward the C terminus (this nomenclature was first defined by Schechter and Berger [1967]); SEC-MALS, size-exclusion chromatography-multiple angle light scattering; SKIE, solvent kinetic isotope effect; SSHB, short strong hydrogen bond; SIHB, short ionic hydrogen bond; TSE, thermal stability enhancement.Article and publication are at http://www.proteinscience.org/cgi

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Section: Y547fsupporting

confidence: 55%

Involvement of DPP‐IV catalytic residues in enzyme–saxagliptin complex formation

et al. 2008

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“…24,25,32,33 Even among serine proteases, it has been reported that the prolyl oligopeptidase family exhibits important dissimilarities in the active site compared to the pancreatic and subtilisin classes of serine proteases. 34 In the case of the prolyl oligopeptidase, non-catalytic histidine imidazole ring-attached protons are engaged in relatively strong hydrogen bonds based on the observation that their downfield chemical shifts are virtually independent of pH up to pH 9.5. 34 Furthermore, while in most situations a LBHB involves a carboxyl group from Asp and Glu residues, 35 our results indicate that this is not always the case.…”

Section: Discussionmentioning

confidence: 99%

“…34 In the case of the prolyl oligopeptidase, non-catalytic histidine imidazole ring-attached protons are engaged in relatively strong hydrogen bonds based on the observation that their downfield chemical shifts are virtually independent of pH up to pH 9.5. 34 Furthermore, while in most situations a LBHB involves a carboxyl group from Asp and Glu residues, 35 our results indicate that this is not always the case. Conclusive assignment of the downfield proton resonance, to the specific His residue as well as to the specific ring nitrogen in our case, is important for the interpretation of experimental results.…”

Section: Discussionmentioning

confidence: 99%

A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A2 and a Transition State Analog Inhibitor

Poi¹,

Tomaszewski²,

Yuan³

et al. 2003

Journal of Molecular Biology

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This work describes in-depth NMR characterization of a unique lowbarrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A 2 (sPLA 2 , from bee venom and bovine pancreas) and a transitionstate analog inhibitor HK32. A downfield proton NMR resonance, at 17 -18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15 N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H 12 of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [ N 12]His. Instead, the downfield resonance arises from H d1 of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(^0.06), and an exchange rate protection factor of . 100 at 285 K and . 40 at 298 K, which are characteristic of a LBHB. The pK a of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp…His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. . 10 kcal/mol).

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“…A preliminary analysis of OpdB function in the context of bacterial growth and protein turnover has been undertaken with Salmonella (13). A preliminary kinetic characterization of the E. coli enzyme illustrated that it cleaved substrates at the C terminus of basic residues, exhibited pronounced substrate inhibition and that the enzyme-substrate interaction was disrupted by high ionic strength, implicating ionic interactions in substrate binding (16,29,32). However, OpdB homologues from prokaryotes have not been extensively characterized, particularly with respect to substrate recognition properties and roles in bacterial virulence.…”

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confidence: 99%

Substrate Recognition Properties of Oligopeptidase B fromSalmonella entericaSerovar Typhimurium

2002

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Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gramnegative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P 1 . While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu 576 and Glu 578 , that define P 1 specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp 460 and Asp 462 , that may be involved in defining P 2 specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.The oligopeptidase B (OpdB; EC 3.4.21.83) subfamily of serine peptidases represents one of two branches of the prolyl oligopeptidase family of serine peptidases (the S9 family, in the nomenclature of Barrett and Rawlings [2]). The archetypical member of this family, prolyl oligopeptidase (POP; EC 3.4.21.26), exclusively hydrolyzes peptide bonds C terminal to proline residues in peptides (31). It has been implicated in the pathophysiology of depression (20) and has attracted pharmaceutical attention, since POP inhibitors have shown potential in the treatment of amnesia (39) and Alzheimer's disease (36). Prolyl oligopeptidase has also served as a model for structural studies of serine oligopeptidases. A 1.4-Å crystal structure analysis of POP recently revealed that an N-terminal regulatory domain, consisting of a seven-bladed ␤-propeller, regulates substrate access to the C-terminal catalytic domain (7) by a gating filter mechanism (8).In contrast to the POPs, the OpdB branch of the POP family has received much less attention. These enzymes demonstrate a trypsin-like substrate specificity, hydrolyzing peptide bonds on the C-terminal side of basic amino acid residues of lowmolecular-mass (Ͻ3 kDa) peptides (17,30,37,41). Of great importance for potential therapeutic applications, OpdB is only found i...

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Low Barrier Hydrogen Bond Is Absent in the Catalytic Triads in the Ground State but Is Present in a Transition-state Complex in the Prolyl Oligopeptidase Family of Serine Proteases

Cited by 29 publications

References 36 publications

Involvement of DPP‐IV catalytic residues in enzyme–saxagliptin complex formation

Involvement of DPP‐IV catalytic residues in enzyme–saxagliptin complex formation

A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A2 and a Transition State Analog Inhibitor

Substrate Recognition Properties of Oligopeptidase B fromSalmonella entericaSerovar Typhimurium

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