Tumor necrosis factor-␣-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC 50 of 70 nM. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC 50 greater that 2 M). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys 184 residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.

The metalloproteinase tumor necrosis factor-␣-converting enzyme (TACE) is involved in the regulation of several key physiological and pathological processes. Therefore, potent and selective synthetic inhibitors are highly sought for the study of the physiological roles of TACE as well as for therapeutic purposes. Because of the high structural similarities between the active site of TACE and those of other related zinc endopeptidases such as disintegrin (ADAMs) and matrix metalloproteinases (MMPs), the design of such tailor-made inhibitors is not trivial. To obtain new insights into this problem, we have used a selective MMP inhibitor as a probe to examine the structural and kinetic effects occurring at the active site of TACE upon inhibition. Specifically, we used the selective MMP mechanism-based inhibitor SB-3CT to characterize the fine structural and electronic differences between the catalytic zinc ions within the active sites of TACE and MMP-2. We show that SB-3CT directly binds the metal ion of TACE as observed before with MMP-2. However, in contrast to MMP-2, the binding mode of SB-3CT to the catalytic zinc ion of TACE is different in the length of the Zn-S(SB-3CT) bond distance and the total effective charge of the catalytic zinc ion. In addition, SB-3CT inhibits TACE in a non-competitive fashion by inducing significant conformational changes in the structure. For MMP-2, SB-3CT behaved as a competitive inhibitor and no significant conformational changes were observed. An examination of the second shell amino acids surrounding the catalytic zinc ion of these enzymes indicated that the active site of TACE is more polar than that of MMP-2 and of other MMPs. On the basis of these results, we propose that although there is a seemingly high structural similarity between TACE and MMP-2, these enzymes are significantly diverse in the electronic and chemical properties within their active sites.

The tumor necrosis factor-alpha-converting enzyme (TACE) is a member of the disintegrin family of metalloproteinases (ADAMs) that plays a central role in the regulated shedding of a host of cell surface proteins. TACE is biosynthesized as a precursor protein with latent proteolytic activity (zymogen). TACE's zymogen inhibition is mediated by its Pro domain, a 197-amino acid region that serves this function as well as aiding in the secretion of this enzyme through the secretory pathway. We have discovered that a conserved "cysteine switch" consensus motif within TACE's Pro domain is, contrary to expectations, not required for maintenance of the inactive precursor state or for the secretion of this metalloproteinase in its functional form. The only role for this motif seems to be in decreasing TACE's susceptibility to proteolytic degradation during its biogenesis and maturation within the secretory pathway. Interestingly, the Pro domain of TACE seems to carry both its inhibitory and secretory functions through the same mechanism: it seems to prevent the Catalytic domain from accessing its native, functional state, resembling the function of true molecular chaperones. Recent evidence suggests that TACE may also be switched out of the active conformation even by small, drug-like molecules such as the synthetic compound SB-3CT. These findings point at the possibility of developing, in the near future, a new generation of antiinflammatory, noncompetitive TACE inhibitors that would exert negative allosteric modulation over the activity of this key enzyme, mediating several inflammatory diseases and certain cancers.