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 K-M(malate) of 0.45 mM and a V-max(malate) of 650 mu mol 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. (C) Academic Press.