Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Dec 20;91(26):12428–12432. doi: 10.1073/pnas.91.26.12428

Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms.

M F White 1, J Vasquez 1, S F Yang 1, J F Kirsch 1
PMCID: PMC45451  PMID: 7809054

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 kcat = 9s-1 per monomer and Km = 12 microM 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 kcat/Km and an increase in Km for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate alpha-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the Km for AdoMet and no change in kcat, suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.

Full text

PDF
12428

Images in this article

12431Fig. 4
on p.12431

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams D. O., Yang S. F. Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc Natl Acad Sci U S A. 1979 Jan;76(1):170–174. doi: 10.1073/pnas.76.1.170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander F. W., Sandmeier E., Mehta P. K., Christen P. Evolutionary relationships among pyridoxal-5'-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families. Eur J Biochem. 1994 Feb 1;219(3):953–960. doi: 10.1111/j.1432-1033.1994.tb18577.x. [DOI] [PubMed] [Google Scholar]
  3. Bleecker A. B., Kenyon W. H., Somerville S. C., Kende H. Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane-1-carboxylate synthase, an enzyme in ethylene biosynthesis. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7755–7759. doi: 10.1073/pnas.83.20.7755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cornforth J. W., Reichard S. A., Talalay P., Carrell H. L., Glusker J. P. Determination of the absolute configuration at the sulfonium center of S-adenosylmethionine. Correlation with the absolute configuration of the diastereomeric S-carboxymethyl-(S)-methionine salts. J Am Chem Soc. 1977 Oct 26;99(22):7292–7300. doi: 10.1021/ja00464a032. [DOI] [PubMed] [Google Scholar]
  5. Cronin V. B., Maras B., Barra D., Doonan S. The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae). Biochem J. 1991 Jul 15;277(Pt 2):335–340. doi: 10.1042/bj2770335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Danishefsky A. T., Onnufer J. J., Petsko G. A., Ringe D. Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase. Biochemistry. 1991 Feb 19;30(7):1980–1985. doi: 10.1021/bi00221a035. [DOI] [PubMed] [Google Scholar]
  7. Goldberg J. M., Swanson R. V., Goodman H. S., Kirsch J. F. The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km. Biochemistry. 1991 Jan 8;30(1):305–312. doi: 10.1021/bi00215a041. [DOI] [PubMed] [Google Scholar]
  8. Graf-Hausner U., Wilson K. J., Christen P. The covalent structure of mitochondrial aspartate aminotransferase from chicken. Identification of segments of the polypeptide chain invariant specifically in the mitochondrial isoenzyme. J Biol Chem. 1983 Jul 25;258(14):8813–8826. [PubMed] [Google Scholar]
  9. Hermann R., Rudolph R., Jaenicke R., Price N. C., Scobbie A. The reconstitution of denatured phosphoglycerate mutase. J Biol Chem. 1983 Sep 25;258(18):11014–11019. [PubMed] [Google Scholar]
  10. Hirel P. H., Schmitter M. J., Dessen P., Fayat G., Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoffman J. L. Chromatographic analysis of the chiral and covalent instability of S-adenosyl-L-methionine. Biochemistry. 1986 Jul 29;25(15):4444–4449. doi: 10.1021/bi00363a041. [DOI] [PubMed] [Google Scholar]
  12. Inoue Y., Kuramitsu S., Inoue K., Kagamiyama H., Hiromi K., Tanase S., Morino Y. Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase. J Biol Chem. 1989 Jun 5;264(16):9673–9681. [PubMed] [Google Scholar]
  13. Jencks W. P. Binding energy, specificity, and enzymic catalysis: the circe effect. Adv Enzymol Relat Areas Mol Biol. 1975;43:219–410. doi: 10.1002/9780470122884.ch4. [DOI] [PubMed] [Google Scholar]
  14. Kende H. Enzymes of ethylene biosynthesis. Plant Physiol. 1989 Sep;91(1):1–4. doi: 10.1104/pp.91.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kondo K., Wakabayashi S., Yagi T., Kagamiyama H. The complete amino acid sequence of aspartate aminotransferase from Escherichia coli: sequence comparison with pig isoenzymes. Biochem Biophys Res Commun. 1984 Jul 18;122(1):62–67. doi: 10.1016/0006-291x(84)90439-x. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li N., Mattoo A. K. Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme. J Biol Chem. 1994 Mar 4;269(9):6908–6917. [PubMed] [Google Scholar]
  18. Lizada M. C., Yang S. F. A simple and sensitive assay for 1-aminocyclopropane-1-carboxylic acid. Anal Biochem. 1979 Nov 15;100(1):140–145. doi: 10.1016/0003-2697(79)90123-4. [DOI] [PubMed] [Google Scholar]
  19. McPhalen C. A., Vincent M. G., Jansonius J. N. X-ray structure refinement and comparison of three forms of mitochondrial aspartate aminotransferase. J Mol Biol. 1992 May 20;225(2):495–517. doi: 10.1016/0022-2836(92)90935-d. [DOI] [PubMed] [Google Scholar]
  20. Mehta P. K., Christen P. Homology of 1-aminocyclopropane-1-carboxylate synthase, 8-amino-7-oxononanoate synthase, 2-amino-6-caprolactam racemase, 2,2-dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and isopenicillin-N-epimerase with aminotransferases. Biochem Biophys Res Commun. 1994 Jan 14;198(1):138–143. doi: 10.1006/bbrc.1994.1020. [DOI] [PubMed] [Google Scholar]
  21. Oeller P. W., Lu M. W., Taylor L. P., Pike D. A., Theologis A. Reversible inhibition of tomato fruit senescence by antisense RNA. Science. 1991 Oct 18;254(5030):437–439. doi: 10.1126/science.1925603. [DOI] [PubMed] [Google Scholar]
  22. Ramalingam K., Lee K. M., Woodard R. W., Bleecker A. B., Kende H. Stereochemical course of the reaction catalyzed by the pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7820–7824. doi: 10.1073/pnas.82.23.7820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rottmann W. H., Peter G. F., Oeller P. W., Keller J. A., Shen N. F., Nagy B. P., Taylor L. P., Campbell A. D., Theologis A. 1-aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence. J Mol Biol. 1991 Dec 20;222(4):937–961. doi: 10.1016/0022-2836(91)90587-v. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sato T., Oeller P. W., Theologis A. The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis. J Biol Chem. 1991 Feb 25;266(6):3752–3759. [PubMed] [Google Scholar]
  26. Satoh S., Yang S. F. Specificity of S-adenosyl-L-methionine in the inactivation and the labeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits. Arch Biochem Biophys. 1989 May 15;271(1):107–112. doi: 10.1016/0003-9861(89)90260-9. [DOI] [PubMed] [Google Scholar]
  27. Van der Straeten D., Van Wiemeersch L., Goodman H. M., Van Montagu M. Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate synthase in tomato. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4859–4863. doi: 10.1073/pnas.87.12.4859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wolfenden R., Sharpless T. K., Allan R. Substrate binding by adenosine deaminase. Specificity, pH dependence, and competition by mercurials. J Biol Chem. 1967 Mar 10;242(5):977–983. [PubMed] [Google Scholar]
  29. Yip W. K., Dong J. G., Kenny J. W., Thompson G. A., Yang S. F. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7930–7934. doi: 10.1073/pnas.87.20.7930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yip W. K., Dong J. G., Yang S. F. Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits. Plant Physiol. 1991 Jan;95(1):251–257. doi: 10.1104/pp.95.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES