Selective replacement of the catalytic zinc of the human stromelysin-1 catalytic domain |
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Authors: | Jaeho Cha M. V. Sørensen Q.-Z. Ye D. S. Auld |
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Affiliation: | (1) Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School and Brigham and Women's Hospital, MA 02115, USA e-mail: david auld@hms.harvard.edu Fax: +1-617-566-3137; Tel.: +1-617-432-1368, US;(2) Parke-Davis Pharmaceutical Research, Warner Lambert Company, Ann Arbor, MI 48105, USA, US |
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Abstract: | We have selectively replaced the catalytic zinc of the catalytic domain of stromelysin-1 (SCD) with other transition metals. Dialysis of the enzyme against 2 mM 1,10-phenanthroline, 20 mM Hepes, pH 7.5 in the presence of 10 mM CaCl2 removes the catalytic zinc, leaving the structural zinc site intact. Dialysis with metal-free buffer followed by the new metal ion replaces the catalytic zinc forming a metal hybrid enzyme. Full incorporation of 1 mol Co2+, Ni2+, or Cd2+/mol enzyme is confirmed by atomic absorption spectrometry while the weaker binding Mn2+ yields a value of 0.4 mol Mn2+/mol enzyme after dialysis against 1 μM Mn2+. The activity of the monozinc enzyme is <10% while its activity is restored upon the addition of zinc and other transition metals. The k cat values for the Co2+, Mn2+, Cd2+, and Ni2+ enzymes are respectively 99%, 54%, 19%, and 17% of the value for the native enzyme, while the respective k cat/K m values are 36%, 29%, 7%, and 16% toward the fluorescent heptapeptide substrate, DnsPLALRAR. The zinc and metal hybrid SCD cleave DnsPLA↓LRAR, and DnsPLE↓LFAR, exclusively at one bond, while DnsPLA↓L↓WAR and DnsPLA↓L↓FAR are cleaved at two positions. The double cleavage of DnsPLALWAR and DnsPLALFAR catalyzed by SCD is in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis is the different cleavage site specificity of the metal hybrids toward the A-L and L-W bonds of the DnsPLALWAR substrate. Thus the k cat values of the Co/Zn hybrid for the cleavage of the A-L bond in the DnsPLALRAR and DnsPLAWAR substrates are 5- and 8-fold greater than those for the Cd/Zn hybrid compared to a 140-fold difference for the corresponding k cat values for the L-W bond cleavage. These results imply that the catalytic metal of SCD is not only involved in catalysis but also influences the substrate specificity of the enzyme. Received: 30 December 1997 / Accepted: 23 February 1998 |
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Keywords: | Matrix metalloproteinase Metal chelating agent Metal hybrid Fluorescent peptide substrate |
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