The Variable Subunit Structure of Lysine-Sensitive Aspartylkinase from Escherichia coli TIR-8

Lysine-sensitive aspartylkinase (AK III) has been purified to homogeneity by the criteria of disc gel electrophoresis at pH 8.3, electrophoresis in sodium dodecyl sulfate, and by sedimentation velocity. It has been shown to be composed of similar subunits of 48,000 ± 4000 molecular weight by sedimentation equilibrium of reduced, carboxymethylated enzyme in 6 M guanidine hydrochloride and by gel electrophoresis in 0.1% sodium dodecyl sulfate. Equilibrium sedimentation, analytical G-200 Sephadex chromatography, and S_20,w plus D_20,W determinations have shown that AK III may be obtained in two forms differing in quaternary structure. One form is a fairly stable dimer of about 100,000 molecular weight, S_20,w = 6.6 S. The second form appears to be in a dimer-tetramer equilibrium and at higher protein concentration behaves as a tetramer of about 200,000 molecular weight, S_20,w = 10.1 S. The partial specific volume of the protein determined by equilibrium sedimentation in D₂O is 0.746 cm³/g, The was determined by dry weight to be 3.60. The specific activity of the enzyme increases during log-phase growth and reaches a sharp maximum at the onset of stationary phase. The enzyme purified from late stationary phase exhibits a high uv absorbancy below 280 nm. Lysine addition to native enzyme causes a difference spectrum with a peak at 294 nm. Titration of the native enzyme at 294 nm exhibits an apparent cooperative transition with the half-maximal change occurring at 0.2 mM lysine.