Roles of cysteinyl residues of phosphoribulokinase as examined by site-directed mutagenesis.
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 (Cys16 and Cys55) 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, Cys244 and Cys250, 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 Km 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 Km for ATP and a 4-8-fold increase in the Km for ribulose 5-phosphate. These results are consistent with moderate facilitation of catalysis by Cys55 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 Ser16 mutant protein to a sulfhydryl reagent supports earlier suggestions that Cys16 is the initial target of the oxidative deactivation process.
Milanez
S
S
Protein Engineering and Molecular Mutagenesis Program, Oak Ridge National Laboratory, University of Tennessee-Oak Ridge, Tennessee 37831-8077.
Mural
R J
RJ
Hartman
F C
FC
D001483:Base Sequence; D015153:Blotting, Western; D003545:Cysteine / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology
D004247:DNA; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D003545:Cysteine; D005033:Ethylmaleimide