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 par ticipates in the coordination of Zn2+ in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substitute d with Ala, Lys or Asp by site-specific mutagenesis. Wildtype proMMP-3, pro MMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escheri chia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type pr oMMP-3 (consisting of the propeptide and catalytic domains) was quantitativ ely converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercur ic acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistan t to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa s pecies. In contrast, treatment with plasmin quantitatively converted the wi ld-type and the three mutant proMMP-3 moieties into the corresponding 24-kD a MMP-3 moieties. Biospecific interaction analysis revealed comparable affi nity for binding to plasminogen of wild-type and mutant proMMP-3 (K-a of 2. 6-6.3 x 10(6) M-1) or MMP-3 (K-a of 33-58 x 10(6) M-1) moieties. The affini ty 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 x 10(6 ) M-1) or MMP-3 (K-a of 37-72 x 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 t owards scu-PA under conditions where this was quantitatively cleaved by wil d-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive towa rd a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the act ivity of wild-type MMP-3. Taken together, these data suggest that Glu202 pl ays a crucial role in the enzymatic activity of MMP-3.