Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103, and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 Received November 28, 1989; Revised Manuscript Received February 16, 1990 abstract: The mechanisms of activation of the precursor of human matrix metalloproteinase 3 (proMMP-3/prostromelysin) by proteinases and (4-aminophenyl) mercuric acetate (APMA) were investigated by kinetic and sequence analyses. Incubation of proMMP-3 with neutrophil elastase, plasma kallikrein, plasmin, or chymotrypsin at 37 C resulted in the formation of MMP-3 of Afr=

45000 by cleaving of the His82-Phe83 bond. Sincethis bond is unlikely to be cleavedby these proteinases it was postulated that an initial attack of an activator proteinase on proMMP-3 creates an intermediate form, which is then processed to a more stable form of MT= 45 000. To test this hypothesis proMMP-3 was incubated with these serine proteinases under conditions that minimize the action of MMP-3. This led to the accumulation of major intermediates of Mr= 53 000 and two minor forms of MT= 49 000 and 47 000. The 53 000 Mr intermediate generated by human neutrophil elastase resulted from cleavage of the Val35-Arg36 bond, whereas plasma kallikrein cleaved the Arg36-Arg37 and Lys38-Asp39 bonds and chymotrypsin the Phe34-Val35 bond, all of which are located near the middle of the propeptide. Conversion of these intermediates to the fully active 45 000 Mr form of MMP-3 resulted from a bimolecular reaction of the intermediates. A similar short-lived intermediate of Mr= 46 000 generatedby APMA was a result of the intramolecular cleavage of the Glu68-Val69 bond, and it was then converted to a stable MMP-3 of Mr=

45000 by a intermolecular reaction of MMP-3. However, MMP-3 failed to activate proMMP-3. These results indicate the removal of the NH2-terminal 34-38 residues by proteinases or 68 residues by APMA is the crucial step for activation of proMMP-3. This initial processing of the propeptide allows the His82-Phe83 bond, which is hindered from proteolysis in native proMMP-3, to be correctly oriented for cleavage by activated intermediates, thereby producing stable 45 000 Mr MMP-3. This stepwise activation process allows proMMP-3 to be activated by various proteinases with distinct substrate specificities.