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 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 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 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 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.
Arza
B
B
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
De Maeyer
M
M
Félez
J
J
Collen
D
D
Lijnen
H R
HR
D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D005341:Fibrinolysin / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D001224:Aspartic Acid; D018698:Glutamic Acid; C010024:4-aminophenylmercuriacetate; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D019278:Matrix Metalloproteinase 3; D015032:Zinc; D008239:Lysine; D000409:Alanine; D010662:Phenylmercuric Acetate