Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate.
نویسندگان
چکیده
The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the alpha-helical surface loop, the so-called 'lid' of HLL comprised of residues 86-93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the 'open' conformation in triacetin, with the alpha-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.
منابع مشابه
The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase.
The importance of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent. The Trp89 mutants displayed an altered chain length specificity towards triglycer...
متن کاملImpact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability.
Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T(d)=74.4 degrees C whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degrees C, respectively, demonstrating significant contribution of the above T...
متن کاملConformational lability of lipases observed in the absence of an oil-water interface: crystallographic studies of enzymes from the fungi Humicola lanuginosa and Rhizopus delemar.
Considerable controversy exists regarding the exact nature of the molecular mechanism of interfacial activation, a process by which most lipases achieve maximum catalytic activity upon adsorption to an oil water interface. X-ray crystallographic studies show that lipases contain buried active centers and that displacements of entire secondary structure elements, or "lids," take place when the e...
متن کاملSurfactant enhanced lipase containing films characterized by confocal laser scanning microscopy.
Confocal laser scanning microscopy (CLSM) in combination with a fluorescently labeling enzyme dye, LavaPurple™, was demonstrated as a technique for the visualization of Thermomyces (Humicola) lanuginosa lipase (LIP(HLL)) and Candida antarctica lipase A (LIP(CA)) within a transparent latex coating. Addition of Teric Surfactants (C(16) non-ionic Teric 475, 1.8% (w/w) or C(10) non-ionic Teric 460,...
متن کاملIsolation and Characterization of a New Thermoalkalophilic Lipase from Soil Bacteria
Lipases are diversified enzymes in their properties and substrate specificity, which make them attractive tools for various industrial applications. In this study, an alkaline thermostable lipase producing bacteria were isolated from soil of different regions of Isfahan province (Iran) and its lipase was purified by ammonium sulfate precipitation and ion exchange chromatography. To select a the...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Biochimica et biophysica acta
دوره 1702 2 شماره
صفحات -
تاریخ انتشار 2004