Spectroscopic and kinetic studies of lipases solubilized in reverse micelles.
The conformation and activity of three different lipases have been studied in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. In the case of human pancreatic lipase, the conformation of the polypeptide chain--as judged from far-UV circular dichroism measurements--is only slightly altered after the enzyme is transferred from a bulk aqueous solution into the microenvironment of reverse micelles. Significant spectral changes in the near-UV circular dichroism and fluorescence spectrum indicate, however, that the solvation of aromatic amino acid side chains is considerably different in reverse micelles. Conversely, the circular dichroism spectra of the lipases from Candida rugosa and Pseudomonas sp. are considerably different in reverse micelles, compared with the spectra in aqueous solution, indicating that both enzymes loose the native structure at the water/AOT/oil interface. Bound substrate and/or product can prevent this denaturation. While Pseudomonas sp. and human pancreatic lipase are inhibited by tetrahydrolipstatin (THL), the lipase from Candida rugosa is not. These data, together with additional activity and inhibition data, indicate that the micellar microenvironment accentuates the difference between the different enzymes in terms of the relation structure/activity.
Walde
P
P
Institut für Polymere, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Han
D
D
Luisi
P L
PL
D000818:Animals; D002175:Candida / Q000201:enzymology; D002942:Circular Dichroism; D004143:Dioctyl Sulfosuccinic Acid; D006801:Humans; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008823:Micelles; D000077403:Orlistat; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013501:Surface-Active Agents; D013552:Swine
D007783:Lactones; D008823:Micelles; D013501:Surface-Active Agents; D004143:Dioctyl Sulfosuccinic Acid; D000077403:Orlistat; D008049:Lipase