Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-[(bromoacetyl)amino]pentitol 1,5-bisphosphate.
2-[(Bromoacetyl)amino]pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum [Fraij, B., & Hartman, F. C. (1982) J. Biol. Chem. 257, 3501-3505]. The enzyme has now been inactivated with a 14C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln- Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser - Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors; D055598:Chemical Phenomena; D002621:Chemistry; D010105:Oxygenases / Q000037:antagonists & inhibitors; D010428:Pentosephosphates; D010446:Peptide Fragments / Q000302:isolation & purification*; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors
C033883:2-bromoacetylaminopentitol 1,5-bisphosphate; D000345:Affinity Labels; D000596:Amino Acids; D001426:Bacterial Proteins; D010428:Pentosephosphates; D010446:Peptide Fragments; D010105:Oxygenases; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase