Characterization of the regulatory thioredoxin site of phosphoribulokinase.
Phosphoribulokinase is light-regulated via thioredoxin by reversible oxidation/reduction of sulfhydryl/disulfide groups. To identify the cysteinyl residues that are involved in regulation, the S-carboxymethyl labeling patterns of the fully reduced (active) and oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced, [14C]carboxymethylated enzyme contained four labeled peptides, all of which were purified and sequenced by Edman degradation. If the enzyme was oxidized by 5,5'-dithiobis-(2-nitrobenzoic acid) prior to carboxymethylation and tryptic digestion, only two labeled peptides were observed, thereby revealing the identity of the regulatory cysteines as Cys-16 and Cys-55. The former was previously implicated as part of the nucleotide-binding domain of the active site (Porter, M.A., and Hartman, F.C. (1986) Biochemistry 25, 7314-7318), a conclusion reinforced by the present observation that the sequence around the Cys-16 is similar to a consensus sequence of ATP-binding sites from a number of proteins of diverse phylogenetic origin (Higgins, C.F., Hiles, I.D., Salmond, G.P.C., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W., and Hermondson, M. (1986) Nature 323, 448-450). The regulatory disulfide of phosphoribulokinase was found to be intrasubunit based on the stoichiometry of the oxidation and the failure to resolve oxidized and reduced enzyme by gel filtration under dissociation conditions.
Porter
M A
MA
Biology Division, Oak Ridge National Laboratory, Tennessee 37830.
Stringer
C D
CD
Hartman
F C
FC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003553:Cystine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D013879:Thioredoxins / Q000378:metabolism*; D014357:Trypsin
D001426:Bacterial Proteins; D010446:Peptide Fragments; D003553:Cystine; D013879:Thioredoxins; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D014357:Trypsin