Spin-labeling studies of urea-treated leucine aminopeptidase.
1. Leucine aminopeptidase (EC 3-4-11-1) from bovine eye lens was spin-labeled at the most reactive thiol groups with 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-1-oxyl. 2. Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B approximately 4:1 was estimated. In B a hydrophobic environment of label was observed. 3. Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B') with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B' shows a time-dependent increase of its extent at the expense of B and A' (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+. b) Mn2+ complexed with the protein is partly released producting hydrated Mn2+. c) After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B approximately 4:1 even after long incubation. 4. 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002729:Chloromercuribenzoates; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology; D007931:Leucyl Aminopeptidase / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D013997:Time Factors; D014508:Urea; D015032:Zinc / Q000494:pharmacology
D002729:Chloromercuribenzoates; D013113:Spin Labels; D008345:Manganese; D014508:Urea; D007931:Leucyl Aminopeptidase; D015032:Zinc