Comparison of inactivation and conformational changes of aminoacylase during guanidinium chloride denaturation.
The inactivation and unfolding of aminoacylase (EC 3.5.1.14) during denaturation by different concentrations of guanidinium chloride (GuHCl) have been compared. A marked decrease in enzyme activity is already evident at low GuHCl concentrations before significant unfolding of the enzyme molecule, as monitored by fluorescence, ultraviolet difference absorption and CD measurement. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of aminoacylase during denaturation by GuHCl. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction kinetics were found to be a monophasic first-order reaction. The kinetics of the unfolding were a bisphasic process consisting of two first-order reactions. At lower GuHCl concentration (< 1.0 M), the enzyme activity was stripped at a high rate whereas its conformation was only slightly affected. At 1.0 M GuHCl, the inactivation rate of the enzyme was much faster than the unfolding rate. At higher GuHCl concentrations (> 1.0 M), the inactivation rate was too fast to be measured by conventional dynamic methods, whereas the unfolding remained as a bisphasic process with the fast reaction accruing very fast and the slow reaction occurring at a measurable rate. The results suggest that active sites of aminoacylase containing Zn2+ ions are situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.
Wang
H R
HR
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, People's Republic of China.
Zhang
T
T
Zhou
H M
HM
D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D000581:Amidohydrolases; C019672:aminoacylase I; D019791:Guanidine