Substrate Specificity Differences between Recombinant Rat Testes Endopeptidase EC 3.4.24.15 and the Native Brain Enzyme

Lew, Rebecca A.; Hey, Nina J.; Tetaz, T.; Glucksman, Marc J.; Roberts, James L.; Smith, A. Ian

doi:10.1006/bbrc.1995.1569

Cited by 27 publications

(23 citation statements)

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“…For example, the potentiation of bradykinin-induced hypotension by cFP-AAF-pAB led Genden & Molineaux (1991) to propose a significant role for EP 24.15 in the turnover of the vasodilator peptide in the circulation. Subsequent studies have shown that cFP-AAY-pAB not only slows bradykinin breakdown, but also blocks the conversion of AI to AII (Yang et al, 1994;Telford et al, 1995), a peptide cleavage not catalyzed by EP 24.15 in vitro (Chu & Orlowski, 1985;Lew et al, 1995). These effects of cFP-AAY-pAB were equivalent to, and not additive with, the effects of ACE inhibitors (Yang et al, 1994;Telford et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo

Lew

Tomoda

Evans

et al. 1996

British J Pharmacology

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1 The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB, K,= 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP-Ala-Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala-Tyr bond of cFP-AAY-pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP-AAY-pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. ACE. Injection of 1 mg kg-' inhibitor resulted in plasma concentrations at 10 s of 23.5 ,uM (cFP-AAYpAB) and 18.0 giM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 1251I-labelled inhibitor revealed that 80-85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25-30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4 These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide-based inhibitors such as cFP-AAY-pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non-peptide, must be developed, for which these peptide-based inhibitors may serve as useful molecular templates.

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Section: Discussionmentioning

confidence: 99%

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo

Lew

Tomoda

Evans

et al. 1996

British J Pharmacology

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“…Thimet oligopeptidase (TOP) is a 78‐kDa endopeptidase (EC 3.4.24.15) that hydrolyzes important neuropeptides including bradykinin, neurotensin, somatostatin, and gonadotropin‐releasing hormone [1–6]. The primary sequence of TOP places it within the class of zinc metalloendopeptidases that includes neurolysin, neutral endopeptidase, and angiotensin‐converting enzyme [7,8].…”

Section: Introductionmentioning

confidence: 99%

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

Sigman

Edwards

Pabon³

et al. 2003

FEBS Letters

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Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clari¢es the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK a of 5.7 and a basic limb with pK a of 8.8. The acidic limb can be attributed to protonation of a residue a¡ecting k cat while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.

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“…The specific and effective cysteine peptidase inhibitor E-64 was shown not to affect EP24.15 activity (12,15), and partial inhibition was only observed at relatively high concentrations in a yeast homologue of EP24.15 (16). Inhibition by thiol-modifying agents such as iodoacetate, iodoacetamide, and N-ethylmaleimide vary from virtually zero to 100%, and inhibition by these agents has been shown to be a time-dependent process (17).…”

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Thiol Activation of Endopeptidase EC 3.4.24.15

Shrimpton¹,

Glucksman²,

Lew³

et al. 1997

Journal of Biological Chemistry

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Endopeptidase EC 3.4.24.15 (EP24.15) is a thermolysin-like metalloendopeptidase involved in the regulated metabolism of a number of neuropeptides. Unlike other thermolysin-like peptidases EP24.15 displays a unique thiol activation, a mechanism that is not clearly understood. In this study we show that both recombinant and tissue-derived EP24.15 are activated up to 8-fold by low concentrations (0.1 mM) of dithiothreitol. Additionally, under non-reducing conditions, recombinant and native EP24.15 forms multimers that can be returned to the monomeric form by reduction. We have also shown that competitive inhibitor binding occurs only to the monomeric form, which indicates that catalytic site access is restricted in the multimeric forms. Through systematic site-directed mutagenesis we have identified that cysteine residues 246, 253, and possibly 248 are involved in the formation of these multimers. Furthermore, both a double mutant (C246S/C253S) and a triple mutant (C246S/C248S/C253S) are fully active in the absence of reducing agents, as measured by both inhibitor binding and hydrolysis. The formation and disruption of disulfide bonds involving these cysteine residues may be a mechanism by which EP24.15 activity is regulated through changes in intra-and extracellular redox potential. Endopeptidase EC 3.4.24.15 (EP24.15) 1 is a 75-kDa neutral metalloendopeptidase that cleaves on the C terminus side of specific hydrophobic residues in substrates of less than 17 amino acids (1, 2). EP24.15 is thought to be involved in the regulated metabolism of a number of neuropeptides including gonadotrophin-releasing hormone, bradykinin, and neurotensin and has more recently been implicated in the aberrant processing of the amyloid precursor protein in Alzheimer's disease (3, 4). EP24.15 is widely distributed in cells and tissues throughout the body, predominantly as a soluble form constituting about 80% of the total activity and as a minor membrane-associated form accounting for the remainder of the activity (5). High levels of EP24.15 activity have been localized, both catalytically and immunohistochemically, primarily to the brain, pituitary, and testis with lower levels in other tissues such as the liver, kidney, spleen, and lung (6).Typical of metalloendopeptidases EP24.15 is inhibited by metal ion chelators and can be reactivated by divalent cations such as Zn 2ϩ or Mn 2ϩ. Like many of the thermolysin-like neutral metalloendopeptidases, the protein's active site contains a zinc ion, which participates in the catalytic process (7) and has the classical HEXXH motif (8) typical of zinc metalloproteases (9). However, unlike the other thermolysin-like metallopeptidases such as angiotensin-converting enzyme, endothelin-converting enzyme, neutral endopeptidase, and the closely related endopeptidase EC 3.4.24.16 (EP24.16), EP24.15 displays a unique thiol activation. EP24.15 is activated by low levels of thiol-containing reducing agents (Ͻ0.5 mM DTT) and inhibited by high concentrations (Ͼ5 mM DTT) (1, 10 -12). Inhibition has been ...

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Substrate Specificity Differences between Recombinant Rat Testes Endopeptidase EC 3.4.24.15 and the Native Brain Enzyme

Cited by 27 publications

References 0 publications

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo

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

Thiol Activation of Endopeptidase EC 3.4.24.15

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