The pyruvate-aspartic semialdehyde condensing enzyme of Escherichia coli.

The pyruvate-aspartic semialdehyde condensing enzyme, which occurs at the branch point in the aspartic acid family of amino acids leading to diaminopimelic acid and lysine biosynthesis, has been purified 5000-fold from crude extracts of Escherichia coli W. The protein has been shown to be homogeneous by polyacrylamide gel electrophoresis and bears a net negative charge in the pH range 6.0 to 9.2. Amino acid analyses indicate the total absence of methionine. The Stokes radius of the condensing enzyme was determined from its elution volume on a Sephadex G-200 column, the sedimentation constant from sucrose density gradient centrifugation, and the partial specific volume from its amino acid composition. These parameters were calculated as: 43 A, 7.1 (s~~,~), and 0.724 cc per g, respectively. The molecular weight of the protein was determined as 134,000 daltons, and its frictional ratio as 1.26. The reaction catalyzed by the condensing enzyme is complex and forms a labile heterocyclic product that has been tentatively identified as dihydrodipicolinic acid. That the condensing enzyme functions in carbon-carbon bond formation by forming an imine linkage with one of its substrates has been shown by the following: (a) there is irreversible loss of enzymatic activity upon the addition of sodium borohydride only in the presence of pyruvate; (b) titration of substrate amounts of purified protein with radioactive pyruvate in the presence of sodium borohydride indicates the presence of four binding sites per protein molecule; (c) acid hydrolysis of protein derivatized with 14C-pyruvate by the borohydride treatment shows the presence of a lysine derivative that has been identified as E-N-(l-carboxyethyl)-L-lysine; (d) the protein catalyzes proton exchange between tritiated pyruvate and water that exhibits first order kinetics and is dependent on protein concentration. A possible role of the enzyme as a nucleophilic catalyst is discussed in connection with its similarity to &aminolevulinic acid dehydratase.