title: Effects of high concentration of salts on the esterase activity and structure of a kiwifruit peptidase, actinidain.
abstract: Effects of salts on the activity and stability of actinidain were examined. With increasing salt concentration up to 0.5 M, the activity (kcat/Km) for N-alpha-Cbz-L-lysine p-nitrophenyl ester decreased to 40% of that in the absence of salt. The inhibitor constant Ki of LiCl, NaCl, and KCl was 0.16-0.43 M. With 3 M KCl and NaCl, the specificity constant kcat/Km recovered to 110 and 75%, respectively. No re-activation was observed with LiCl. The inhibition and re-activation were dependent on the changes in both Km and kcat, whereas no CD change was observed. The tryptophan fluorescence of actinidain was not affected by 0-0.5 M salt, but a considerable decrease in its intensity was observed with increasing salt concentration from 0.5 to 3.0 M. These results suggest that the inhibition observed with the lower salt concentration (<0.5 M) is due to attenuation of the electrostatic interaction between the enzyme and substrate, and the higher concentration (0.5-3.0 M) induces structural change in the states of tryptophan residues, which is associated with the re-activation. Actinidain keeps considerably high activity and stability even in the presence of 3 M salts.
authors.affiliation: Department of Biotechnological Science, Kinki University, Nishimitani, Kinokawa, Wakayama 649-6493.
authors.forename: Koichi
authors.initials: K
authors.lastname: Morimoto
authors.forename: Erino
authors.initials: E
authors.lastname: Furuta
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hashimoto
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D029042:Actinidia / Q000187:drug effects / Q000201:enzymology*; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D004950:Esterases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D018021:Lithium Chloride / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D012492:Salts / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D012492:Salts; D012965:Sodium Chloride; D011189:Potassium Chloride; D004950:Esterases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; C018745:actinidain; D018021:Lithium Chloride