Biochemical and molecular characterization of fumarase from plants: purification and characterization of the enzyme--cloning, sequencing, and expression of the gene.
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.
Behal
R H
RH
Department of Botany, Iowa State University, Ames 50011, USA. bbehal@iastate.edu
Oliver
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005649:Fumarate Hydratase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017343:Genes, Plant; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum; D013552:Swine
D011994:Recombinant Proteins; D005649:Fumarate Hydratase