Evidence for essential lysyl residues in ribulosebisphosphate carboxylase by use of the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.
A previous study from our laboratory suggested that 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is an affinity label for spinach ribulosebisphosphate carboxylase. To identify the essential residues that react with the reagent we have isolated and characterized the labeled peptides that are present in tryptic digests of inactivated enzyme but lacking in digests of the substrate-protected enzyme. Peptides representing two sites of modification have been obtained from the inactivated carboxylase. Both sites of reaction have been identified as lysyl residues based on the conversion of the derivatives to free lysine by oxidation with sodium metaperiodate. Sodium dodecyl sulfate-gel electrophoretic experiments show that both essential lysyl residues are contained within the large subunit of ribulosebisphosphate carboxylase. In addition to lysyl residues, sulfhydryl groups of the carboxylase are also modified, but their modification seems to play little role in the inactivation process. The carboxylase modified in the presence of substrate contains sulfhydryl derivatives but is essentially lacking in lysyl derivatives. By comparing the profiles from ion exchange chromatography of labeled peptides in digests of inactivated and substrate-protected enzyme, we conclude that the same sulfhydryl groups are modified in the absence and presence of substrate.
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002074:Butanones; D002262:Carboxy-Lyases / Q000032:analysis*; D008239:Lysine / Q000032:analysis; D009943:Organophosphorus Compounds; D010446:Peptide Fragments / Q000032:analysis; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis*
D000345:Affinity Labels; D000596:Amino Acids; D002074:Butanones; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine