Effect of mutagenic replacement of the carboxyl terminal amino acid, val124, on the properties and regeneration of bovine pancreatic ribonuclease A.
An important role of C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) in the formation of the three-dimensional structure was previously implied. In this study, we replaced the C-terminal amino acid, Val124, with amino acid residues with different properties by site-directed mutagenesis. The recombinant mutant enzymes were purified and subjected to a refolding study after being converted to a fully reduced and denatured state. There was a significant difference among the mutant enzymes in the rate of recovery of the activity when air oxidation was performed: the rate decreased in the order of V124E, V124L, V124G, V124K, V124A, and V124W. On the other hand, the recovery rates for all the mutant RNase A in the presence of GSH and GSSG were almost the same. The recovered activity of V124E after 24 h incubation reached approximately 90% of that of the wild type enzyme, followed by V124L 80%, V124A and V124W 65%, and V124K and V124G 50%. The duration of the initial lag phase became shorter in the order of V124W, V124A, V124K or V124G, V124E, or V124L. The results imply that the C-terminal amino acid significantly influences the formation of correct disulfide bonds during the refolding process and that the hydrophobic interaction of Val124 is important for efficient packing of the RNase A molecule.
Fujii
T
T
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Doi
Y
Y
Ueno
H
H
Hayashi
R
R
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*; D014633:Valine
D012259:Ribonuclease, Pancreatic; D005978:Glutathione; D014633:Valine