Detergent-induced conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy

Detergent (pentaoxyethylene octyl ether, C8E5)-induced conformational chang es of Humicola lanuginosa lipase (HLL) were investigated by stationary and time-resolved fluorescence intensity and anisotropy measurements. Activatio n of HLL is characterized by opening of a surface loop (the "lid") residing directly over the enzyme active site. The interaction of HLL with C8E5 inc reases fluorescence intensities, prolongs fluorescence lifetimes, and decre ases the values of steady-slate anisotropy, residual anisotropy, and the sh ort rotational correlation time. Based on these data, we propose the follow ing model. Already below critical micellar concentration (CMC) the detergen t can intercalate into the active site accommodating cleft, while the lid r emains closed. Occupation of the cleft by C8E5 also blocks the entry of the monomeric substrate, and inhibition of catalytic activity at [C8E5] less t han or equal to CMC is evident. At a threshold concentration close to CMC t he cooperativity of the hydrophobicity-driven binding of C8E5 to the lipase increases because of an increase in the number of C8E5 molecules present i n the premicellar nucleates on the hydrophobic surface of HLL. These aggreg ates 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 C8E5 and the "open" conformation of HLL bec omes stable.