Selective replacement of the catalytic zinc of the human stromelysin-1 catalytic domain

Jaeho Cha, Marianne V. Sørensen, Qizhuang Ye, David S. Auld

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 DnsPLAL- WAR 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.

Original languageEnglish (US)
Pages (from-to)353-359
Number of pages7
JournalJournal of Biological Inorganic Chemistry
Volume3
Issue number4
DOIs
StatePublished - Aug 1998
Externally publishedYes

Fingerprint

Matrix Metalloproteinase 3
Zinc
Catalytic Domain
Metals
Enzymes
Dialysis
Substrates
Catalysis
Transition metals
Matrix Metalloproteinase 7
Atomic absorption spectrometry
Substrate Specificity
Metal ions
Spectrum Analysis
Buffers
Ions

Keywords

  • Fluorescent peptide substrate
  • Matrix metalloproteinase
  • Metal chelating agent
  • Metal hybrid

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Selective replacement of the catalytic zinc of the human stromelysin-1 catalytic domain. / Cha, Jaeho; Sørensen, Marianne V.; Ye, Qizhuang; Auld, David S.

In: Journal of Biological Inorganic Chemistry, Vol. 3, No. 4, 08.1998, p. 353-359.

Research output: Contribution to journalArticle

Cha, Jaeho ; Sørensen, Marianne V. ; Ye, Qizhuang ; Auld, David S. / Selective replacement of the catalytic zinc of the human stromelysin-1 catalytic domain. In: Journal of Biological Inorganic Chemistry. 1998 ; Vol. 3, No. 4. pp. 353-359.
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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 DnsPLAL- WAR 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.",
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AU - Ye, Qizhuang

AU - Auld, David S.

PY - 1998/8

Y1 - 1998/8

N2 - 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 DnsPLAL- WAR 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.

AB - 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 DnsPLAL- WAR 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.

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KW - Metal hybrid

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