Site-directed mutagenesis of the ATP-binding site in spinach rubisco activase
Shen, Jennie Bih-Jien
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Permalink
https://hdl.handle.net/2142/19253
Description
Title
Site-directed mutagenesis of the ATP-binding site in spinach rubisco activase
Author(s)
Shen, Jennie Bih-Jien
Issue Date
1989
Doctoral Committee Chair(s)
Ogren, William L.
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Plant Physiology
Language
eng
Abstract
Spinach rubisco activase has two isoforms derived from alternate splicing with the larger polypeptide containing an additional 4 kD C-terminal domain. Both isoforms, with molecular masses of 41 kD and 45 kD, were synthesized and purified from the transformed E. coli containing the cDNAs. A significant difference in the kinetics of the ATP response between the two cloned isoforms was observed. The 45 kD polypeptide exhibited sigmoidal kinetics for both rubisco activase and ATPase activities, while the activities of the 41 kD isoform appeared to follow standard Michaelis-Menten kinetics. These observations indicate that the extra C-domain in the 45 kD isoform may play an essential role in controlling the enzyme activity and/or substrate (ATP) binding.
Site-directed mutagenesis was performed to create single amino acid changes in the putative ATP-binding site of rubisco activase. The protein accumulation levels in the transformed E. coli were significantly different with single amino acid substitutions. The mutant proteins of spinach rubisco activase were purified from E. coli by the same purification procedures as the wild-type. The mutation of Lys169 to Arg, Ile or Thr abolished both the rubisco activase and ATPase activities, and the mutant proteins lost the ability to bind ATP. Lys165 could not be substituted by Ala or Arg since the resultant proteins were insoluble. When Lys165 was replaced by Met in both isoforms, the mutant proteins were soluble and possessed reduced rubisco activase and ATPase activities. The mutation of Gln167 to Lys inactivated rubisco activase activity. When Gln167 was changed to Glu, rubisco activase activity increased in the 41 kD isoform, but retained wild-type activity in the 45 kD isoform. The ATPase activity did not parallel the changes in rubisco activase activity. Rather, a higher ratio of rubisco activase to ATPase activity was caused by the Glu167 mutation in both isoforms. Attempts to replace Ser170 with Thr were not successful. Replacement of Ser170 with Pro created an inactive protein. The role of Cys314 was not determined, but the mutation of Cys314 to Ser in the 45 kD isoform reduced both rubisco activase and ATPase activities.
The results indicate that single amino acid substitutions in the ATP-binding domain were sufficient to eliminate or alter the rubisco activase and ATPase specific activities. The changes in these two activities of the mutant proteins were not directly correlated, indicating that the rubisco activase and ATPase activities are not tightly coupled.
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