Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions.
The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 381-436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn2+ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.
Zhang
Y X
YX
Department of Chemistry, Capitol University of Medical, Sciences, Beijing, China.
Yan
S L
SL
Zhou
H M
HM
D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism
D014270:Trifluoroethanol; D000581:Amidohydrolases; C019672:aminoacylase I; D015032:Zinc