Effect of metal ions on the fluorescence of leucine aminopeptidase and its dansyl-peptide substrates.
The effect of Cu(II), Ni(II), Zn(II), Mg(II), and Mn(II) on the fluorescence of porcine kidney cytosol leucine aminopeptidase and three of its dansyl(Dns) peptide substrates, Leu-Gly-NHNH-Dns, Leu-Gly-NH(CH2)2NH-Dns, and Leu-Gly-NH(CH2)6NH-Dns, has been investigated. These five metal ions were chosen for study because each binds to the regulatory metal binding site of leucine aminopeptidase. Since the binding is relatively weak, kinetic studies of the different metalloderivatives of the enzyme are normally carried out in the presence of large molar excesses of these metal ions that can potentially affect both the enzyme and substrate. The fluorescence of all of the dansyl-peptides, as well as several other dansyl species, is quenched by Ni(II) and Cu(II), but not by Mg(II), Mn(II), or Zn(II). The absorption spectra of these dansyl substrates are also perturbed by Ni(II) and Cu(II). The rate at which maximal quenching for some dansyl species is attained after mixing with Ni(II) and Cu(II) is slow and the quenching is reversed on addition of EDTA. These results indicate that the quenching is the result of complex formation between the fluorophores and these metal ions. The association constants for the metal complexes have been determined from Stern-Volmer plots. In addition to complex formation, Ni(II) and Cu(II) cause the degradation of Leu-Gly-NHNH-Dns through a two step mechanism involving loss of dansic acid. Ni(II) and Cu(II) also partially quench the fluorescence of leucine aminopeptidase through contact with its surface accessible Trp residues. These observations indicate that care must be taken in stopped flow fluorescence studies of reactions between this enzyme and its dansyl substrates to avoid adverse effects brought about by Ni(II) and Cu(II).
Lin
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Department of Chemistry, Florida State University, Tallahassee 32306-3015.
Van Wart
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HE
D000818:Animals; D003300:Copper / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D003619:Dansyl Compounds; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology*; D009532:Nickel / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D003619:Dansyl Compounds; D008670:Metals; D003300:Copper; D009532:Nickel; D007931:Leucyl Aminopeptidase