Roles of cysteinyl residues of phosphoribulokinase as examined by site-directed mutagenesis
- ORNL, Oak Ridge, TN (United States) Univ. of Tennessee, Knoxville, TN (United States)
The Calvin Cycle enzyme phosphoribulokinase is activated in higher plants by the reversible reduction of a disulfide bond, which is located at the active site. To determine the possible contribution of the two regulatory residues (Cys[sup 16] and Cys[sup 55]) to catalysis, site-directed mutagenesis has been used to replace each of them in the spinach enzyme with serine or alanine. The only other cysteinyl residues of the kinase, Cys[sup 244] and Cys[sup 250], were also replaced individually by serine or alanine. A comparison of specific activities of native and mutant enzymes reveals that substitutions at positions 244 or 250 are inconsequential. The position 16 mutants retain 45-90% of the wild-type activity and display normal K[sub m] values for both ATP and ribulose 5-phosphate. In contrast, substitution at position 55 results in 85-95% loss of wild-type activity, with less than a 2-fold increase in the K[sub m] for ATP and a 4-8-fold increase in the K[sub m] for ribulose 5-phosphate. These results are consistent with moderate facilitation of catalysis by Cys[sup 55] and demonstrate that the other three cysteinyl residues do not contribute significantly either to structure or catalysis. The enhanced stability, relative to wild-type enzyme, of the Ser[sup 16] mutant protein to a sulfhydryl reagent supports earlier suggestions that Cys[sup 16] is the initial target of the oxidative deactivation process.
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 5366625
- Journal Information:
- Journal of Biological Chemistry; (United States), Journal Name: Journal of Biological Chemistry; (United States) Vol. 266:16; ISSN JBCHA3; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
Similar Records
Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16
Selective modification of phosphoribulokinase cysteines
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
CARBOHYDRATES
CARBOXYLIC ACIDS
CYSTEINE
ENZYMES
KETONES
MOLECULAR STRUCTURE
MONOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PENTOSES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
RIBULOSE
SACCHARIDES
STRUCTURE-ACTIVITY RELATIONSHIPS
THIOLS
TRANSFERASES