In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca2+ and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62 kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56 kDa, 52 kDa and 50 kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62 kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50 kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients kcat, Km and kcat/Km were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower kcat and kcat/Km values as well as a slightly higher Ki value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.
- promatrix metalloproteinase-2