Kinetics of reactivation during refolding of guanidine-denatured pancreatic ribonuclease A.
The method aforementioned (Liu, W. and Tsou, C.L. (1987) Biochim. Biophys. Acta 916, 455-464) for the study of the kinetics of irreversible modification of enzyme activity has been applied to the reactivation of guanidine-denatured ribonuclease A, by following the hydrolysis of cyclic CMP during refolding upon diluting a guanidine-denatured enzyme with a substrate-containing buffer. Appropriate equations have been derived to deal with the kinetics of the substrate reaction during the course of activation, while the product formed, 3'CMP, is a competitive inhibitor. When the overall process consists of multiple first-order reactions, the individual rate constants could be obtained by suitable semilogarithmic plots. Moreover, in certain cases, it can be distinguished from the shapes of the plots, whether the overall process consists of parallel or consecutive first-order reactions. The kinetics for the reactivation reaction has been compared to that for the refolding of the substrate binding site, as indicated by complex formation with the competitive inhibitor, 2'CMP, and for the refolding of the molecule as a whole. At pH 6.0 and 25 degrees C, only monophasic first-order reactions could be detected by manual mixing for both the reactivation and the refolding processes. At lower temperatures (0-10 degrees C), both processes consist of two first-order reactions. In all cases, the same rate constants have been obtained for the refolding and reactivation reactions.
Liu
W
W
Laboratory of Molecular Enzymology, Institute of Biophysics, Academia Sinica, Beijing, China.
Tsou
C L
CL
D000465:Algorithms; D000818:Animals; D002417:Cattle; D003080:Cold Temperature; D003563:Cyclic CMP / Q000378:metabolism; D003568:Cytidine Monophosphate / Q000378:metabolism; D004789:Enzyme Activation; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions
D006146:Guanidines; D012996:Solutions; D003563:Cyclic CMP; D003568:Cytidine Monophosphate; D019791:Guanidine