title: Catalytic nonessentiality of an active-site cysteinyl residue of phosphoribulokinase.
abstract: The activity of the Calvin cycle enzyme phosphoribulokinase is coupled to photosynthetic electron transport by reversible oxidation/reduction mediated by thioredoxin-f. Previous studies have shown that one of the regulatory sulfhydryl groups, that of Cys-16, is positioned at the nucleotide-binding domain of the active site. To determine if oxidative deactivation of the kinase reflects catalytic essentiality of Cys-16, the methylation of spinach phosphoribulokinase by methyl-4-nitrobenzenesulfonate has been examined. Methylation of the kinase results in a 50% loss of the initial activity relative to controls. The suppression of kcat is accompanied by a 6-fold increase in the Km for ATP, without change in the Km for ribulose 5-phosphate. The insensitivity of the modified enzyme, in contrast to the native, to iodoacetate and 5,5'-dithiobis(2-nitrobenzoate) indicates that Cys-16 is a site of methylation. This supposition is verified independently by peptide mapping and Edman degradation subsequent to S-carboxymethylation with [14C]iodoacetate of the methylated kinase. Retention of significant enzymatic activity after complete modification of Cys-16 with the small, uncharged methyl moiety demonstrates that this active-site residue is not essential for catalysis.
authors.affiliation: Biology Division, Oak Ridge National Laboratory, Tennessee.
authors.forename: M A
authors.initials: MA
authors.lastname: Porter
authors.forename: F C
authors.initials: FC
authors.lastname: Hartman
mesh_terms: D001557:Benzenesulfonates / Q000494:pharmacology; D001665:Binding Sites; D003545:Cysteine; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D010449:Peptide Mapping; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology; D014357:Trypsin
chemical_list: D001557:Benzenesulfonates; C010233:methyl-4-nitrobenzenesulfonate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D014357:Trypsin; D003545:Cysteine; D004229:Dithiothreitol