Exploration of the function of a regulatory sulfhydryl of phosphoribulokinase from spinach.
Phosphoribulokinase from spinach is deactivated by reversible oxidation of Cys16 and Cys55 to an intrasubunit disulfide. Both residues have been assigned to the nucleotide-binding domain of the active site. Clearly, Cys16 does not play a significant role in catalysis, as complete methylation of this residue decreases kcat by only 50%. With methylated enzyme as the starting material, modification by 2-nitro-5-thiocyanobenzoate was used to probe the function of Cys55. The reagent rapidly inactivates methylated kinase, and activity is fully restored by dithiothreitol treatment. ATP, and ribulose 5-phosphate retard inactivation. The stoichiometry of incorporation indicates that only one site per subunit undergoes cyanylation. Mapping of tryptic digests demonstrates that Cys55 is selectively labeled by the reagent. The low level of activity observed after modification of Cys55 by the sterically unobtrusive cyano group suggests that Cys55 could play a facilitative role in catalysis; alternatively, slight reorientation of other catalytic groups as a consequence of cyanylation of Cys55 could account for the inactivation. In either event, major conformational changes need not be invoked to account for the loss of kinase activity concomitant with regulatory oxidation.
Porter
M A
MA
Biology Division, Oak Ridge National Laboratory 37831-8077.
Hartman
F C
FC
D004220:Disulfides / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013861:Thiocyanates / Q000494:pharmacology; D014675:Vegetables / Q000201:enzymology*
D004220:Disulfides; D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D013861:Thiocyanates; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; C008658:2-nitro-5-thiocyanobenzoic acid