Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

Journal Article · Mon Oct 01 00:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.20.7930· OSTI ID:5584167

Yip, Wing-Kin; Dong, Jian-Guo; Yang, S F ^[1]; Kenny, J W; Thompson, G A ^[2]

---

1. Univ. of California, Davis (USA)
2. Calgene Inc., Davis, CA (USA)

The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase 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 NaB{sup 3}H{sub 4} or Ado({sup 14}C)Met. Peptide sequencing of both {sup 3}H- and {sup 14}C-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 {sup 3}H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the {sup 14}C-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 ({sup 14}C)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.

• OSTI ID: 5584167
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 87:20; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ETHYLENE
BIOSYNTHESIS
GENES
DNA SEQUENCING
PEPTIDES
AMINO ACID SEQUENCE
CARBON 14 COMPOUNDS
LYSINE
MASS SPECTROSCOPY
TRITIUM COMPOUNDS
ALKENES
AMINO ACIDS
CARBOXYLIC ACIDS
HYDROCARBONS
HYDROGEN COMPOUNDS
LABELLED COMPOUNDS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
SPECTROSCOPY
STRUCTURAL CHEMICAL ANALYSIS
SYNTHESIS
550201* - Biochemistry- Tracer Techniques