Dihydrodipicolinate synthase, the first enzyme unque to lysine biosynthesis in higher plants, was The biosynthesis of lysine first utilizes the common pathway for the aspartic-family amino acids including lysine, threonine, and methionine, followed by a pathway specific to lysine biosynthesis (5). Since many, though not all, of the enzymes involved in the lysine-specific pathway have been isolated, higher plants are supposed to synthesize lysine via the diaminopimelate pathway (5). In our effort to clarify the regulation of lysine biosynthesis in wheat, we first reported the effects of lysine analogs and aspartate-derived amino acids on growth and AK' (EC. 2.7.2.4) activity in wheat cell suspension cultures (26). The fact that lysine inhibits wheat AK, the first enzyme in the common pathway for aspartate-family amino acids, but not growth of wheat cells, indicates that there should be a more potent site of feedback-inhibition by lysine in the lysine-specific pathway. DHDPS (EC 4.2.1.52), the first enzyme uniquely associated with lysine biosynthesis, catalyzes the condensation of L-ASA and pyruvate. This enzyme activity has been detected in several plants (6,(13)(14)(15)(16)25), and has been found to be localized in the chloroplasts (24). DHDPS from higher plants is feedback-inhibited by low concentrations of lysine (15,16,25) for the enzyme from Escherichia coli (28). For the E. coli enzyme, it is also reported that an enzyme-pyruvate complex exists by Schiff base formation (21) and that the inhibition by lysine is competitive with respect to ASA (20). The kinetic mechanism and the mode of inhibition, however, have not been explored in detail. In view of the potential importance of DHDPS in the regulation of lysine biosynthesis, we studied its properties and kinetics using highly purified enzyme preparations from wheat suspension cultures.
MATERILS AND METHODSPlant Material. Suspension cultures of wheat (Triticum aestivum var Chinese Spring), established as described previously (26), were grown on a gyrotory shaker at 100 rpm, 25°C, in the dark. Liquid LS medium (12) supplemented with 10 Mm, 2,4-D, in a total volume of 75 ml, were used in 300-ml flasks. Inoculum weight was 2.0 to 2.5 g fresh weight per flask. Subcultures were carried out at 3-week intervals.Chemicals. Lysine analogs were obtained from the following sources: AEC, DHL, Na-acetyl-L-lysine, Nf-acetyl-L-lysine, and L-lysine hydroxamate were from Sigma Chemical Co.; Ne methyl-L-lysine from Aldrich Chemical Co.; THL from Calbiochem-Behring Corp; and cis and trans isomers of DL-lysyne (A4-lysine) dihydrochloride, DL-lysyne dihydrochloride (26), DL-4-oxalysine dihydrochloride, and DL-homolysine monohydrochloride were synthesized as described previously (26).Butyl-Toyopearl 650 M was obtained from Toyo Soda Manufacturing Co., Tokyo. Sephadex G-25 M, PD-I0 columns, DEAESepharose CL-6B, Phenyl-Superose HR 10/10, Mono Q HR 5/ 5, Superose-12 HR 10/30, and a FPLC system were from Pharmacia, Inc. Protein standards for HPLC were obtained from Oriental...
