Characterization of an active-site peptide modified by glyoxylate and pyridoxal phosphate from spinach ribulosebisphosphate carboxylase/oxygenase.
Activated ribulosebisphosphate carboxylase/oxygenase from spinach was treated with glyoxylate plus or minus the transition-state analog, carboxyarabinitol bisphosphate, or the inactive enzyme with pyridoxal phosphate plus or minus the substrate, ribulose bisphosphate. Covalently modified adducts with glyoxylate or pyridoxal phosphate were formed following reduction with sodium borohydride. The derivatized enzymes were carboxymethylated and digested with trypsin; the labeled peptides which were unique to the unprotected samples were purified by ion-exchange chromatography and gel filtration. Both glyoxylate and pyridoxal phosphate were associated with only one major peptide, which in each case was subjected to amino acid analysis and sequencing. The sequence was -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys(Cm)-Thr-Ile-Lys-Lys*-Pro-Lys-, with both reagents exhibiting specificity for the same lysine residue as indicated by the asterisk. This peptide is identical to that previously isolated from spinach carboxylase labeled with either of two different phosphorylated affinity reagents and homologous to one from Rhodospirillum rubrum carboxylase modified by pyridoxal phosphate. The species invariance of this lysine residue, number 175, and the substantial conservation of adjacent sequence support the probability for a functional role in catalysis of the lysyl epsilon-amino group.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D006038:Glyoxylates / Q000378:metabolism / Q000494:pharmacology; D010105:Oxygenases / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*
D000596:Amino Acids; D006038:Glyoxylates; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D010105:Oxygenases; D012273:Ribulose-Bisphosphate Carboxylase; C031150:glyoxylic acid