Detergent-induced conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy.
Detergent (pentaoxyethylene octyl ether, C(8)E(5))-induced conformational changes of Humicola lanuginosa lipase (HLL) were investigated by stationary and time-resolved fluorescence intensity and anisotropy measurements. Activation of HLL is characterized by opening of a surface loop (the "lid") residing directly over the enzyme active site. The interaction of HLL with C(8)E(5) increases fluorescence intensities, prolongs fluorescence lifetimes, and decreases the values of steady-state anisotropy, residual anisotropy, and the short rotational correlation time. Based on these data, we propose the following model. Already below critical micellar concentration (CMC) the detergent can intercalate into the active site accommodating cleft, while the lid remains closed. Occupation of the cleft by C(8)E(5) also blocks the entry of the monomeric substrate, and inhibition of catalytic activity at [C(8)E(5)] less than or equal to CMC is evident. At a threshold concentration close to CMC the cooperativity of the hydrophobicity-driven binding of C(8)E(5) to the lipase increases because of an increase in the number of C(8)E(5) molecules present in the premicellar nucleates on the hydrophobic surface of HLL. These aggregates contacting the lipase should have long enough residence times to allow the lid to open completely and expose the hydrophobic cleft. Concomitantly, the cleft becomes filled with C(8)E(5) and the "open" conformation of HLL becomes stable.
Jutila
A
A
Helsinki Biophysics and Biomembrane Group, Department of Medical Chemistry, Institute of Biomedicine, University of Helsinki, Helsinki, Finland.
Zhu
K
K
Patkar
S A
SA
Vind
J
J
Svendsen
A
A
Kinnunen
P K
PK
D001665:Binding Sites; D003902:Detergents; D005454:Fluorescence Polarization; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D003904:Mitosporic Fungi / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors
D003902:Detergents; D011994:Recombinant Proteins; D008049:Lipase