Purification and characterization of cysteine synthases from Citrullus vulgaris

Abstract

Two forms of cysteine synthase from Citrullus vulgaris seedlings were purified ca 2000-fold to homogeneity, respectively, using both conventional and affinity chromatographic methods. Both isoenzymes were found to have the same M r s of58 000, and SDS-PAGE showed that they contained two identical subunits with M r s of 29 000, respectively. Amino acid analysis indicated that isoenzymes A and B have almost similar amino acid compositions, except for the number of cysteine and methionine residues. The K m value of isoenzyme A is 2.6 mM for O-acetyl- l-serine (OAS) and 36 μM for sulphide, while that of isoenzyme B is 1.5 mM for OAS and 33 μM for sulphide. Data on the substrate specificity show that both isoenzymes catalyse the formation of β-(pyrazol-1-yl)- l-alanine and β-(3-amino-1,2,4-triazol-1-yl)- l-alanine, although only isoenzyme A catalyses the formation of β-cyano- l-alanine. Several properties of the purified cysteine synthase isoenzymes are described.