PURIFICATION AND CHARACTERIZATION OF DIHYDRODIPICOLINATE SYNTHASE FROM PEA

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a co mbination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicol inate synthase from a plant source. The pea dihydrodipicolinate syntha se has an apparent molecular weight of 127,000 and is composed of thre e identical subunits of 43,000 as determined by gel filtration and cro ss-linking experiments. The trimeric quaternary structure resembles th e trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phos phogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference conc erns the methionine content: dihydrodipicolinate synthase from pea con tains 22 moles of methionine residue per mole of native protein, contr ary to the E. coli enzyme, which does not contain this amino acid at a ll. Dihydrodipicolinate synthase from pea is highly specific for the s ubstrates pyruvate and L-aspartate-beta-semialdehyde; it follows Micha elis-Menten kinetics for both substrates. The pyruvate and L-aspartate -beta-semialdehyde have Michaelis constant values of 1.70 and 0.40 mil limolar, respectively. L-Lysine, S-(2-aminoethyl)-L-cysteine, and L-al pha-(2-aminoethoxyvinyl)glycine are strong allosteric inhibitors of th e enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, re spectively. The inhibition by L-lysine and L-alpha-(2-aminoethoxyvinyl )glycine is noncompetitive towards L-aspartate-beta-semialdehyde, wher eas S-(2-aminoethyl)-L-cysteine inhibits dihydrodipicolinate. Furtherm ore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid ( 1.2 millimolar) activates dihydrodipicolinate synthase from pea by a f actor of 1.4 and 1.2, respectively. This is the first reported activat ion process found for dihydrodipicolinate synthase.