EXPRESSION OF APPLE 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE IN ESCHERICHIA-COLI - KINETIC CHARACTERIZATION OF WILD-TYPE AND ACTIVE-SITE MUTANT FORMS

Malcolm F White, J VASQUEZ, S F YANG, J F KIRSCH

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72 Citations (Scopus)

Abstract

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple ACC synthase was overexpressed in Escherichia coli (3 mg/liter) and purified to near homogeneity. A continuous assay was developed by coupling the ACC synthase reaction to the deamination of 5'-methylthioadenosine by adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Aspergillus oryzae. The enzyme is dimeric, with k(cat) = 9 s(-1) per monomer and K-m = 12 mu M for AdoMet. The pyridoxal phosphate-binding site of ACC synthase appears to be highly homologous to that of aspartate aminotransferase, suggesting similar roles for corresponding residues. Site-directed mutagenesis of Lys-273, Arg-407, and Tyr-233 (corresponding to residues 258, 386, and 225 in aspartate aminotransferase) and kinetic analyses of the mutants confirms their importance in the ACC synthase mechanism. The Lys-273 to Ala mutant has no detectable activity, supporting the identification of this residue as the base catalyzing C-alpha proton abstraction, Mutation of Arg-407 to Lys results in a precipitous drop in k(cat)/K-m and an increase in K-m for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate cu-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the K-m for AdoMet and no change in k(cat), suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.

Original languageEnglish
Pages (from-to)12428-12432
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number26
Publication statusPublished - 20 Dec 1994

Keywords

  • ADENOSINE DEAMINASE
  • PYRIDOXAL PHOSPHATE
  • SITE-DIRECTED MUTAGENESIS
  • CONTINUOUS ASSAY
  • MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE
  • ADENOSYL-L-METHIONINE
  • AMINO-ACID-SEQUENCE
  • ETHYLENE BIOSYNTHESIS
  • MONOCLONAL-ANTIBODIES
  • MESSENGER-RNA
  • TOMATO
  • PURIFICATION
  • ENZYME
  • FRUIT

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