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

TitleCharacterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase
Authors
Issue Date1990
PublisherNational Academy of Sciences. The Journal's web site is located at http://www.pnas.org
Citation
Proceedings Of The National Academy Of Sciences Of The United States Of America, 1990, v. 87 n. 20, p. 7930-7934 How to Cite?
AbstractThe 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.
Persistent Identifierhttp://hdl.handle.net/10722/178483
ISSN
2015 Impact Factor: 9.423
2015 SCImago Journal Rankings: 6.883
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorYip, WKen_US
dc.contributor.authorDong, JGen_US
dc.contributor.authorKenny, JWen_US
dc.contributor.authorThompson, GAen_US
dc.contributor.authorYang, SFen_US
dc.date.accessioned2012-12-19T09:47:57Z-
dc.date.available2012-12-19T09:47:57Z-
dc.date.issued1990en_US
dc.identifier.citationProceedings Of The National Academy Of Sciences Of The United States Of America, 1990, v. 87 n. 20, p. 7930-7934en_US
dc.identifier.issn0027-8424en_US
dc.identifier.urihttp://hdl.handle.net/10722/178483-
dc.description.abstractThe 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.en_US
dc.languageengen_US
dc.publisherNational Academy of Sciences. The Journal's web site is located at http://www.pnas.orgen_US
dc.relation.ispartofProceedings of the National Academy of Sciences of the United States of Americaen_US
dc.subject.meshAmino Acid Sequenceen_US
dc.subject.meshBinding Sitesen_US
dc.subject.meshFruiten_US
dc.subject.meshLyases - Genetics - Metabolismen_US
dc.subject.meshLysine - Analogs & Derivatives - Analysisen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshPeptide Mappingen_US
dc.subject.meshPlants - Enzymologyen_US
dc.subject.meshPyridoxal - Analogs & Derivatives - Analysisen_US
dc.subject.meshSequence Homology, Nucleic Aciden_US
dc.subject.meshTrypsinen_US
dc.titleCharacterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthaseen_US
dc.typeArticleen_US
dc.identifier.emailYip, WK: wkyip@hkucc.hku.hken_US
dc.identifier.authorityYip, WK=rp00833en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1073/pnas.87.20.7930-
dc.identifier.pmid2122449-
dc.identifier.scopuseid_2-s2.0-0025155485en_US
dc.identifier.volume87en_US
dc.identifier.issue20en_US
dc.identifier.spage7930en_US
dc.identifier.epage7934en_US
dc.identifier.isiWOS:A1990ED61200029-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridYip, WK=7102784428en_US
dc.identifier.scopusauthoridDong, JG=37042970700en_US
dc.identifier.scopusauthoridKenny, JW=7202807763en_US
dc.identifier.scopusauthoridThompson, GA=7403077227en_US
dc.identifier.scopusauthoridYang, SF=8857896100en_US

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