Fluorescence study of fungal lipase from Humicola lanuginosa

Time-resolved and steady-state fluorescence quenching measurements have bee n performed to study two different conformations of the fungal lipase from Humicola lanuginosa. The intrinsic fluorescence of tryptophan Trp89 residue , located in the 'lid' region; has been used as a probe for the dynamics of protein. The native ('closed-lid') form of the enzyme has been found to de cay as a triple exponential with time constants and relative contributions of 5.4 ns (74.3%), 2.2 ns (20.4%) and 0.4 ns (5.3%). A comparison of recove red decay parameters obtained for native and mutated H. lanuginosa lipase s hows that Trp89 contributes about 61% to the class of emitting species with the lifetime of 5.4 ns: The fluorescence quenching data show that three ou t of four tryptophans (i.e., 117, 221 and 260 residues) within H. lanuginos a. lipase are totally quenchable by acrylamide while completely inaccessibl e to iodide. On the contrary, the Trp89 residue is available for both quenc hers. Using steady-state iodide fluorescence quenching data and the fluores cence-quenching-resolved-spectra (FQRS) method, the total emission spectrum of the native Lipase has been decomposed into two spectral components. One of them, unquenchable by iodide, has a maximum of fluorescence emission at 330 nm and the second one, exposed to the solvent, emits at 338. nm. The r esolved spectrum of the redder component corresponds to the Trp89 residue, which participates in about 65% of the total H. lanuginosa emission: The dy namic Stern-Volmer quenching constants calculated for both native ('closed- lid') and inhibited ('open-lid') lipase are 2.71 and 4.49; M-1, respectivel y. The values obtained indicate that Trp89 is not deeply: buried in the pro tein matrix. Our results suggest that distinct configurations of fungal Lip ase can be monitored using the fluorescence of the Trp89 residue located in the 'lid'-helix which participate's in an interfacial activation of the en zyme. (C) 1998 Elsevier Science S.A. All rights reserved.