Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis.
The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)
Lin
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WY
Department of Chemistry, Florida State University, Tallahassee 32306.
Van Wart
H E
HE
D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D003619:Dansyl Compounds / Q000378:metabolism*; D004735:Energy Transfer; D006868:Hydrolysis; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D008433:Mathematics; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D002268:Carboxypeptidases; D007931:Leucyl Aminopeptidase; D043422:Carboxypeptidases A