Critical role of glutamic acid 202 in the enzymatic activity of stromelysin‐1 (MMP‐3)

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn 2+ in matrix metalloproteinase‐3 (MMP‐3 or stromelysin‐1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site‐specific mutagenesis. Wild‐type proMMP‐3, proMMP‐3(E202A), proMMP‐3(E202K) and proMMP‐3(E202D) were expressed in Escherichia coli  and purified to apparent homogeneity. Whereas 33‐kDa wild‐type proMMP‐3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24‐kDa active MMP‐3 by treatment with p‐aminophenyl‐mercuric acetate (APMA), proMMP‐3(E202A) and proMMP‐3 (E202K) were fully resistant to APMA and proMMP‐3 (E202D) was quantitatively converted into a 14‐kDa species. In contrast, treatment with plasmin quantitatively converted the wild‐type and the three mutant proMMP‐3 moieties into the corresponding 24‐kDa MMP‐3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild‐type and mutant proMMP‐3 ( K a of 2.6–6.3 × 10 6   m −1 ) or MMP‐3 ( K a of 33–58 × 10 6   m −1 ) moieties. The affinity for binding to single‐chain urokinase‐type plasminogen activator (scu‐PA) was also similar for wild‐type and mutant proMMP‐3 ( K a of 5.0–6.9 × 10 6   m −1 ) or MMP‐3 ( K a of 37–72 × 10 6   m −1 ) moieties. However, MMP‐3(E202A) and MMP‐3(E202K) did not hydrolyze plasminogen whereas MMP‐3(E202D) showed an activity of 20–30% of wild‐type MMP‐3. All three mutants were inactive towards scu‐PA under conditions where this was quantitatively cleaved by wild‐type MMP‐3. Furthermore, MMP‐3(E202A) and MMP‐3(E202K) were inactive toward a fluorogenic substrate and MMP‐3 (E202D) displayed about 15% of the activity of wild‐type MMP‐3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP‐3.