Abstract
The metalloproteinase tumor necrosis factor-alpha-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.
Highlights
The metalloproteinase tumor necrosis factor-␣-converting enzyme (TACE) is involved in the regulation of several key physiological and pathological processes
Based on results discussed below, it seems that SB-3CT interacts with the active sites of TACE and matrix metalloproteinases (MMPs)-2 in entirely distinct ways despite the high sequence and structural similarity of their catalytic domains
SB-3CT was found to inhibit TACE in the low micromolar range with a Ki value of 3.7 Ϯ 1.0 M, which is worse approximately by two orders of magnitude compared with the inhibition of MMP-2 by the same inhibitor
Summary
The catalytic domain of human TACE was expressed using a recombinant baculovirus expression system. This truncate was purified to homogeneity from the culture medium of infected Trichoplussia ni cells as described earlier [7, 13]. The cell pellet was resuspended in 50 mM Tris, pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, and 1% Triton X-100 at a 1:25 ratio of the buffer to the original culture volume. The suspension was centrifuged for 10 min at 15,000 rpm, and the pellet was dissolved in 50 mM Tris, pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, and 0.2% Sarkosyl followed by a 30-min incubation on ice. The supernatant fraction was loaded onto a 5-ml gelatin-Sepharose column (prepacked, Amersham Biosciences), preequilibrated, and washed with dialysis buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 5 mM CaCl2, 10 M ZnCl2, 0.02% Brij).
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