Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate.
We reported earlier (Schloss, J. V., and Hartman, F. C. (1977) Biochem. Biophys. Res. Commun. 77, 230-236) that N-bromoacetylethanolamine phosphate is an affinity label for spinach ribulosebisphosphate carboxylase/oxygenase. We now show inactivation to be correlated directly with the alkylation either of a single lysyl residue (in the presence of Mg2+) or of 2 different cysteinyl residues (in the absence of Mg2+), consistent with the likelihood that these residues are located in the active site region. This proposition is further supported by the demonstration that the residues are protected from alkylation by substrate, a competitive inhibitor, or the transition state analog 2-carboxyribitol bisphosphate. Tryptic peptides that contain the modified residues have been isolated and sequenced. One of the 2 cysteinyl residues that are subject to alkylation is only 3 residues distant in sequence from the lysyl residue modified by bromoacetylethanolamine phosphate. This lysyl residue is identical with 1 of the 2 lysyl residues alkylated by the previously described affinity label, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (Stringer, C. D., and Hartman, F. C. (1978) Biochem. Biophys, Res. Commun. 80, 1043-1048).
D000345:Affinity Labels / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism; D003545:Cysteine; D004983:Ethanolamines / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010105:Oxygenases / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism
D000345:Affinity Labels; D000596:Amino Acids; D004983:Ethanolamines; D009943:Organophosphorus Compounds; C020161:N-bromoacetylethanolamine phosphate; D010105:Oxygenases; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine; D003545:Cysteine