Partial purification and characterization of dihydrodipicolinic acid synthetase from sporulating Bacillus megaterium.
Sporulation of Bacillus megaterium Km (ATCC 13632) was synchronized by a technique employing three 10% transfers. The culture was harvested when 60% of the cells contained spore forms. Dihydrodipicolinic acid synthetase was purified 150-fold by ammonium sulfate fractionation at pH 6.5, heating for 15 min at 45 C at pH 6.0, ammonium sulfate fractionation at pH 6.0, and subsequent chromatography on diethylaminoethyl cellulose. During the final stage of the purification procedure, the enzyme exhibited sensitivity to refrigeration temperatures. The enzyme had a pH optimum of 7.65 in imidazole buffer. The apparent K(m) values were 4.6 x 10(-4) and 5.0 x 10(-4)m for beta-aspartyl semialdehyde and pyruvate, respectively. All attempts to demonstrate cofactor requirements were unsuccessful. Sulfhydryl inhibiting reagents and lysine did not inhibit the enzymatic reaction. The enzyme exhibited maximal thermal resistance at pH 10.5. The thermal stability of the enzyme at 75 C was increased more than 1,800-fold by the addition of 0.3 m pyruvate. The E(a) was 67,300 cal/mole for the thermal denaturation of the enzyme. At 60 C, the DeltaF, DeltaH, and DeltaS values for the thermal denaturation of the enzyme were 22,250, 66,700, and 133 cal per mole per degree, respectively.
D001410:Bacillus megaterium / Q000201:enzymology*; D002021:Buffers; D002848:Chromatography, DEAE-Cellulose; D003470:Culture Media; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles; D008190:Lyases / Q000302:isolation & purification*; D008239:Lysine / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D010848:Picolinic Acids / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis; D013057:Spectrum Analysis; D013170:Spores / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D002021:Buffers; D003470:Culture Media; D007093:Imidazoles; D010848:Picolinic Acids; D011506:Proteins; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D008190:Lyases; D008239:Lysine