Biochemical and molecular characterization of fumarase from plants: purification and characterization of the enzyme--cloning, sequencing, and expression of the gene

Arch Biochem Biophys. 1997 Dec 1;348(1):65-74. doi: 10.1006/abbi.1997.0359.

Abstract

A cDNA EST clone encoding the C-terminal portion of Arabidopsis thaliana fumarase was identified by homology analysis. A fragment of cDNA encoding the N-terminal region of fumarase was amplified from a cDNA library using PCR and cloned. Genomic DNA corresponding to the coding region of fumarase was amplified and cloned. Arabidopsis fumarase was expressed as a chimeric fusion protein and polyclonal antibodies were generated. Fumarase was purified to near-homogeneity (over 600-fold) from etiolated Pisum sativum mitochondria. The identification of fumarase was confirmed by a combination of immunoblot and N-terminal amino acid sequencing. Kinetic analysis of highly purified fumarase yielded a KM(malate) of 0.45 mM and a Vmax(malate) of 650 mumol of fumarate/min/ mg. The pea fumarase was inhibited by the alpha-keto acids pyruvate and alpha-ketoglutarate at low millimolar concentrations. Adenylates were highly inhibitory; the degree of this inhibition was reduced in the presence of Mg2+, suggesting that uncomplexed adenylates are the inhibitory species.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arabidopsis / enzymology*
  • Arabidopsis / genetics
  • Base Sequence
  • Cloning, Molecular
  • Escherichia coli / enzymology
  • Fumarate Hydratase / chemistry
  • Fumarate Hydratase / isolation & purification
  • Fumarate Hydratase / metabolism*
  • Genes, Plant*
  • Humans
  • Kinetics
  • Mitochondria / enzymology
  • Molecular Sequence Data
  • Peas / enzymology*
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Solanum tuberosum
  • Swine

Substances

  • Recombinant Proteins
  • Fumarate Hydratase