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[(4C]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-LeuPro-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 "4C-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.The plant hormone ethylene regulates many aspects of plant growth and development. Adams and Yang (1) have established that ethylene is biosynthesized via the following se-

Turnover of 1-Aminocyclopropane-1-Carboxylic Acid Synthase Protein in Wounded Tomato Fruit Tissue

“…In wounded tomato fruit discs treated with CHI, Kende and Boller (10) (17) showed that the rate of ACC synthase inactivation in vitro depends upon the concentration of AdoMet and that in the presence of AVG, a competitive inhibitor of ACC synthase with respect to AdoMet (7), the half-life of the enzyme activity in vitro increases from 54 to 108 mmn. Later work showed that this AdoMet-dependent inactivation results from an irreversible covalent linkage of the 2-aminobutyrate moiety of AdoMet to the enzyme (18,25).…”

“…Spanu et al (21) 3B) and other previous data (26). It should be noted that both native and AdoMet-inacti-_4 vated ACC synthase proteins are recognized by the monoclonal antibody (17,25). Thus, if the inactivation of ACC synthase by AdoMet represents the first step in its turnover, this inactivated enzyme must be further degraded by a rapid proteolytic process (17).…”

“…The apparent half-life of ACC synthase activity in the tissue, as measured in the presence of CHI, was estimated to be about 40, 25, and 40 min in wounded tomato tissue (10), in auxin-treated mung bean hypocotyls (26), and in elicitortreated tomato cell culture (21), respectively, indicating that the turnover of ACC synthase activity is rapid.…”

Nucleotide Sequence of a cDNA Clone Encoding 1-Aminocyclopropane-1-Carboxylic Acid Synthase from Broccoli

“…Two enzymes, ACC synthase and ACC oxidase, are responsible for ethylene biosynthesis from its precursor S-adenosyl Met (Yang and Hoffman, 1984), and blocking the synthesis of these enzymes in antisense RNA experiments results in reduced ethylene synthesis (Hamilton et al, 1990;Oeller et al, 1991). Functional identification of cDNA clones encoding ACC synthase has been achieved by expression studies in Escherichia coli and yeast and by sequence comparisons to peptides purified from ACC synthase protein (Sato and Theologis, 1989;Yip et al, 1990;Dong et al, 1991).…”

“…T h e predicted peptides of the broccoli, tomato, and apple clones were aligned (Devereux et al, 1984), and regions of high identity were found to correspond to conserved regions of other ACC synthases (Park et al, 1992). The pbroc3 cDNA contained a region with 83% amino acid identity (100% similarity based on conservative changes) with the peptide sequence identified as the active site of ACC synthase (Yip et al, 1990). Broccoli (Brassica oleraceae L. var ltalica cv Shogun).…”