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 ofArabidopsis thalianafumarase 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.Arabidopsisfumarase was expressed as a chimeric fusion protein and polyclonal antibodies were generated. Fumarase was purified to near-homogeneity (over 600-fold) from etiolatedPisum sativummitochondria. The identification of fumarase was confirmed by a combination of immunoblot and N-terminal amino acid sequencing. Kinetic analysis of highly purified fumarase yielded aKM(malate) of 0.45 mM and aVmax(malate) of 650 μmol of fumarate/min/mg. The pea fumarase was inhibited by the α-keto acids pyruvate and α-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.