Roles of the propeptide and metal ions in the folding and stability of the catalytic domain of stromelysin (matrix metalloproteinase 3).

Matrix metalloproteinases (MMPs) are an unique class of zinc metalloproteases in that 12 A from the catalytic zinc site is a second zinc site, the function of which has yet to be determined. In the pro form, the protease is inactive. Here we show that the heat-induced autocatalytic activation of pro to mature MMP3 is bimolecular. Further, the process is modulated by a low-affinity zinc. A mechanism is proposed by which the second zinc site may act as an enzymatic activator for the mature protease. A method for preparing completely metal-free protein is described. Surprisingly, there is a much more dramatic structural change between the apo and holo forms of the mature protein than there is between apo and holo proprotein. Apo mature MMP3 appears to form a native-like stable intermediate structure in which one or more of the tryptophan side chains is more solvent-exposed than in the holo form. Apo MMP3 is remarkably stable to thermal unfolding as monitored by CD; thus the metal ions do not appear to significantly stabilize the secondary structure of the catalytic domain. The apo mature MMP3 intermediate can be unfolded with heat, subsequently refolded, and reactivated by addition of zinc and calcium. Thus for MMP3, unlike subtilisin or alpha-lytic protease, the propeptide is not required for protein folding in a timely fashion and the role of intramolecular chaperone is not a universal one for the propeptides of proteases.