L-LYSIN - ALPHA-KETOGLUTARATE AMINOTRANSFERASE .2. PURIFICATION CRYSTALLIZATION AND PROPERTIES

The preparation of crystalline L-lysine-α- ketoglutarate aminotransferase from Achromobacter liquidum is described. The enzyme is homogeneous by the criteria of ultracentrifugation, free-boundary electrophoresis, and disc gel electrophoresis. The molecular weight is 116,000, and 2 moles of pyridoxal 5′-phosphate are bound per mole of holoenzyme. The enzymeexhibits absorption maxima at 415 and 340 mμ. No appreciable spectral change was observed on varying pH. Addition of an amino donor to the enzyme produced a decrease in the absorbance at 415 mμ and an increase in that in the 340-mμ region with concomitant shift of the maximum from 340 to 330 mμ. The spectrum of the enzyme was not influenced by addition of α-ketoglutarate. Incubation of the enzyme with L-lysine in the presence of a high concentration of phosphate gave an inactive form of the enzyme (semiapoenzyme) which could be reactivated with pyridoxal 5′-phosphate. The absorption spectrum of semiapoenzyme does not have an absorption maximum at 415 mμ, but does have one at 340 mμ. This inactive form of enzyme contains 1 mole of pyridoxal 5′-phosphate. L-Lysine-α-ketoglutarate aminotransferase exhibits optimal activity at pH 8.3-8.5; it is stable over the pH range 6.0-7.5. It catalyzes transfer of the terminal amino groups of L-lysine and L-ornithine to α-ketoglutarate which is the exclusive amino acceptor. The following Michaelis constants were determined: L-lysine (2.8 × 10−3 m), α-ketoglutarate (5.0 × 10−4 m), and pyridoxal 5′-phosphate (3.57 × 10−7 M). © 1968, American Chemical Society. All rights reserved.