title: Steady-state kinetics of hydrolysis of dansyl-peptide substrates by leucine aminopeptidase.
abstract: Stopped-flow fluorescence experiments have been carried out at 23 degrees C to study the hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] and Leu-Gly-NH(CH2)2NH-Dns by porcine kidney cytosol leucine aminopeptidase (LAP). Experiments have been performed with LAP species containing Mg(II), Mn(II), Ni(II), Cu(II), Zn(II), and no metal ion at the regulatory metal binding site. The fluorescence changes observed on hydrolysis of these dansyl substrates by LAP arise from changes in the concentration of substrate. Several kinetic relationships have been developed that allow the steady-state kinetic parameters for these reactions to be determined from the stopped-flow fluorescence traces. When any of the five metal ions are bound at the regulatory site, kcat and KM are both raised to approximately the same extent with the result that the maximum increase observed for kcat/KM is only approximately twofold. The effects of these metal ions on kcat, KM, and kcat/KM observed for these substrates differ markedly from those for less physiologically relevant substrates, such as Leu-p-nitroanilide, that do not have amino acids on both sides of the scissile bond. This suggests that earlier conclusions regarding the effect of the regulatory metal ion on the activity of LAP may have been misleading and casts doubt as to whether the term "regulatory site" has validity in the context of LAP-catalyzed reactions under physiological conditions.
authors.affiliation: Department of Chemistry, Florida State University, Tallahassee 32306.
authors.forename: W Y
authors.initials: WY
authors.lastname: Lin
authors.forename: S H
authors.initials: SH
authors.lastname: Lin
authors.forename: H E
authors.initials: HE
authors.lastname: Van Wart
mesh_terms: D000465:Algorithms; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D003619:Dansyl Compounds / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D013552:Swine
chemical_list: D003619:Dansyl Compounds; D007931:Leucyl Aminopeptidase