Disruption of the cysteine-75 and zinc ion coordination is not sufficient to activate the precursor of human matrix metalloproteinase 3 (stromelysin 1).
Matrix metalloproteinases (MMPs) are activated in vitro from their precursors (proMMPs) by multiple means such as treatment with proteinases, mercurial compounds, chaotropic agents, sodium dodecyl sulfate, HOCl, and heat. The latency of proMMPs is stabilized by intramolecular interaction of the single cysteine residue in the conserved sequence PRCG(V/N)PD of the propeptide and the zinc atom at the active site. The activation of proMMP-1 (interstitial procollagenase) by multiple treatments has been explained by the "cysteine switch" model, in which the disruption of the Cys-Zn interaction is considered to be critical for activation [Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., & VanWart, H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 364-368]. To further test this hypothesis we dissociated the Cys-Zn interaction by specifically modifying Cys-75 of proMMP-3 (prostromelysin 1) with iodoacetamide, (4-aminophenyl)mercuric acetate (APMA), or 5,5'-dithiobis(2-nitrobenzoate) and examined the expression of enzymic activity. The enzymic assays of the modified proMMP-3s against protein and synthetic substrates did not reveal any significant activity. The modified 57-kDa proMMP-3s were stable and did not show spontaneous activation. Activation of the modified proMMP-3s required further treatment with APMA or a proteinase and was accompanied by conversion of the proMMP-3 to a 45-kDa species. Circular dichroism studies of proMMP-3 treated with HgCl2 demonstrated time-dependent conformational changes in proMMP-3 prior to the expression of proteolytic activity and processing of the zymogen to lower molecular weight species. These results indicate that the disruption of the Cys-Zn coordination alone is not sufficient to activate proMMP-3.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421.
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Chen
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Noelken
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Nagase
D000478:Alkylation; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D019278:Matrix Metalloproteinase 3; D008627:Mercuric Chloride / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry*
D013439:Sulfhydryl Reagents; D008627:Mercuric Chloride; C010024:4-aminophenylmercuriacetate; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D015032:Zinc; D003545:Cysteine; D010662:Phenylmercuric Acetate; D007460:Iodoacetamide