title: Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase.
abstract: The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14), the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB3H4 or Ado[14C]Met. HPLC separation of the trypsin digest yielded a single radioactive peptide. Peptide sequencing of both 3H- and 14C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado[14C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.
authors.affiliation: Department of Vegetable Crops, Mann Laboratory, University of California, Davis 95616.
authors.forename: W K
authors.initials: WK
authors.lastname: Yip
authors.forename: J G
authors.initials: JG
authors.lastname: Dong
authors.forename: J W
authors.initials: JW
authors.lastname: Kenny
authors.forename: G A
authors.initials: GA
authors.lastname: Thompson
authors.forename: S F
authors.initials: SF
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005638:Fruit; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010944:Plants / Q000201:enzymology*; D011730:Pyridoxal / Q000031:analogs & derivatives / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin
chemical_list: C017696:epsilon-pyridoxyllysine; D011730:Pyridoxal; D014357:Trypsin; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase; D008239:Lysine