Thiol Activation of Endopeptidase EC 3.4.24.15

Abstract

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 ...

Crystal Structure of Human Thimet Oligopeptidase Provides Insight into Substrate Recognition, Regulation, and Localization

“…Mutation of Cys-682 and Cys-687, on the other hand, does not improve activity, consistent with their location on the backside of the enzyme far away from the channel opening. Mutating Cys-46 is not sufficient to prevent all loss of activity, and it seems clear that other cysteines, most prominently cysteines 246, 248, and 253, also mediate inactivation by multimerization (35,37). It is difficult to imagine how multimerization involving these residues and any of the other surface cysteines would occlude the active site channel, and determining if loss of activity is a steric or a conformational effect awaits further experimentation.…”

“…The enzyme loses activity in the absence of reducing agents, and this inactivation occurs in part by the formation of disulfide-linked multimers, which appear to lose affinity for substrate (35,37). Shrimpton et al (35) suggest that polymerization may provide a mechanism for modulating TOP activity based on the redox characteristics of its environment.…”

“…In addition, Cys-18 and Cys-682 are located in the disordered N and C termini, respectively (not included in the crystal structure), and are, therefore, also likely to be solvent-accessible. In the rat ortholog, cysteines 246, 248, 253, and 682 have been implicated in multimer formation (35,37), and all are among the surface-accessible residues in the crystal structure of the human enzyme. Cysteines at positions 46 and 687 in rat TOP are also thought to be involved in multimer formation.…”

“…To obtain crystals, we used a modified version of thimet oligopeptidase, which begins at residue 16 and has residues 246 and 253 mutated from cysteine to serine. The N terminus was removed because the corresponding residues of the closely related neurolysin were disordered in the crystal structure (49), and the cysteines were mutated to prevent covalent oligomerization of the enzyme (35). Full-length native thimet oligopeptidase (both human and rat orthologs) did not crystallize despite extensive screening.…”

“…Expression of TOP activity is regulated at the level of transcription (32)(33)(34), but activity may also be regulated by posttranslational modification. TOP oligomerizes by intermolecular disulfide bond formation in the absence of reductant, resulting in a decrease or loss of activity, and this modulation by multimerization may be a physiological regulatory mechanism (35)(36)(37). The activity of rat TOP can also be modulated by phosphorylation, and phosphorylated forms of the enzymes have been isolated from cultured cells (38,39).…”