pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15)

Sigman, Jeffrey A.; Edwards, Sarah R.; Pabon, Amanda; Glucksman, Marc J.; Wolfson, Adele J.

doi:10.1016/s0014-5793(03)00548-9

Cited by 14 publications

(25 citation statements)

References 34 publications

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“…These findings, as well as the mechanism proposed earlier [10] and previous data reported in the literature [9], strongly indicate that Cys residues 682 and 687 are involved in protein oligomerization. This involvement would explain why, at 1 mM GSSG concentration, the protein does not oligomerize, and why both Cys 682 and 687 were glutathiolated.…”

Section: Discussionsupporting

confidence: 86%

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

et al. 2012

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Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10–50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.

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Section: Discussionsupporting

confidence: 86%

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

et al. 2012

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“…Relative to the parameters of ZapA on its best substrate, the Michaelis constant ( K m ) and the catalytic efficiency ( k cat ) values for PrtA on our substrate were 2‐ and 100‐fold higher, respectively, suggesting weaker ground‐state stabilization and better positioning of the scissile bond. Comparable activities of metallo‐peptidases on synthetic substrates have been reported for Clostridium hystolyticum collagenase (M26 family) [27] and peptidases in the thimet oligopeptidase (M3) family [28,29]. The Dabcyl–EVYAVES–Edans substrate allowed detection of as low as 1–3 pmoles of enzyme at a substrate concentration of 55 µ m , which is a 3–10‐fold higher sensitivity of detection for PrtA activity than achieved with zymography (10–20 pmoles; not shown).…”

Section: Resultsmentioning

confidence: 90%

Cleavage site analysis of a serralysin‐like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate

et al. 2007

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The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and beta-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1' and P2'. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1' > P2 > P2'. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4'(dimethylamino)phenylazo]benzoyl-EVYAVES-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The approximately 4 x 10(6) M(-1) x s(-1) specificity constant of PrtA (at K(m) approximately 5 x 10(-5) M and k(cat) approximately 2 x 10(2) s(-1)) on this substrate was the highest activity for a serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.

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“…A previous report demonstrated the influence of calcium and other divalent cations activating EP24.15 enzyme activity, in a substrate dependent manner [38] consistent with our data. Another recent study has also shown the influence of calcium on the acid limb of a pH‐dependence activity curve of EP24.15 [39]. Fluorogenic substrates have also been used previously to show the influence of different salts on the EP24.15 activity [40].…”

Section: Discussionmentioning

confidence: 99%

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity

Oliveira¹,

Garrido²,

Rodrigues³

et al. 2005

The FEBS Journal

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The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function. Abbreviations EP24.15; metalloendopeptidase 24.15; G6PD, glucose-6-phosphate dehydrogenase; PFA, paraformaldehyde.

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pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15)

Cited by 14 publications

References 34 publications

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

Cleavage site analysis of a serralysin‐like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity

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