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), Vol. 266:16; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PHOSPHOTRANSFERASES
STRUCTURE-ACTIVITY RELATIONSHIPS
CYSTEINE
MOLECULAR STRUCTURE
RIBULOSE
AMINO ACIDS
CARBOHYDRATES
CARBOXYLIC ACIDS
ENZYMES
KETONES
MONOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PENTOSES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
SACCHARIDES
THIOLS
TRANSFERASES
550200* - Biochemistry