The closed/open model for lipase activation. Addressing intermediate active forms of fungal enzymes by trapping of conformers in water-restricted environments.
The behavior of prototypic fungal lipases in a water-restricted environment has been investigated by exploiting the reported experimental strategy that allows the trapping (freeze-drying) of the enzyme in the conformation present in aqueous solution and to subsequently assay it in nonaqueous media [Mingarro, I., Abad, C., and Braco, L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3308-3312]. We now report, using simple esterification as well as acidolysis (triglycerides as substrates) as nonaqueous model reactions, that the presence of a detergent (n-octyl-beta-glucopyranoside) in the freeze-drying buffer, at concentrations below the critical micellar concentration, generates different catalytically active (kinetically trapped) conformational states of the enzyme. These activated forms exquisitely discriminate between short- and long-chain fatty acids, suggesting that they can be correlated with intermediate conformations of the protein sufficiently open to permit the access of relatively small but not large substrates. Additional data obtained from aqueous solution activity measurements in the presence of detergent revealed that the fungal lipase retains an active conformation induced by high detergent concentration (30 mM) for a long period of time, a 'memory effect', which is stabilized in the absence of a well-defined interface by few detergent molecules. Together these results provide support to a model of lipase action involving several equilibrium states (closed, intermediate, and open), which can be modulated by the composition of the microenvironment, i.e., by the detergent concentration.
Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de València, E-46100 Burjassot, Spain.
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González-Navarro
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Bañó
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Abad
D001203:Ascomycota / Q000201:enzymology; D002021:Buffers; D002175:Candida / Q000201:enzymology; D003902:Detergents; D004789:Enzyme Activation; D004795:Enzyme Stability; D005612:Freeze Drying; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D005848:Geotrichum / Q000201:enzymology; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D008823:Micelles; D008956:Models, Chemical; D011487:Protein Conformation; D012233:Rhizopus / Q000201:enzymology; D012996:Solutions; D013863:Thioglucosides / Q000737:chemistry; D014280:Triglycerides / Q000737:chemistry; D014867:Water
D002021:Buffers; D003902:Detergents; D005656:Fungal Proteins; D008823:Micelles; D012996:Solutions; D013863:Thioglucosides; D014280:Triglycerides; D014867:Water; C042989:n-octyl-beta-D-thioglucopyranoside; D008049:Lipase