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Characterization of glutamate‐1‐semialdehyde aminotransferase of Synechococcus

1991; Wiley; Volume: 202; Issue: 3 Linguagem: Inglês

10.1111/j.1432-1033.1991.tb16429.x

1432-1033

Marvin A. Smith, C. Gamini Kannangara, Bernhard Grimm, Diter von Wettstein,

Polyamine Metabolism and Applications

Synechococcus glutamate‐1‐semialdehyde aminotransferase was expressed in large amounts in transformed cells of Esherichia coli. The resulting purified enzyme has an absorption spectrum characteristic of B 6 ‐containing enzymes and could be converted to the pyridoxal‐phosphate form with excess dioxovalerate (O 2 Val), and back to the pyridoxamine‐phosphate form with diaminovalerate (A 2 Val). Both enzyme forms are similarly active in the conversion of glutamate 1‐semialdehyde (GSA) to 5‐aminolevulinate (ALev), suggesting that A 2 Val and O 2 Val are intermediates. Initial rates of Alev synthesis at various fixed concentrations of GSA followed typical Michaelis‐Menten kinetics ( K m of GSA for the pyridoxamine‐phosphate form of GSA aminotransferase = 12 μM, k cat = 0.23 s −1 ). In submicromolar amounts A 2 Val stimulates ALev synthesis, and in a series of concentrations with various fixed concentrations of GSA, gives a family of parallel lines in Lineweaver‐Burk plots ( K m for A 2 Val = 1.0 μM). On the other hand, O 2 Val gives competitive inhibition of the pyridoxamine‐phosphate form of GSA‐aminotransferase and mixed‐type inhibition of the pyridoxal‐phosphate form ( K i for O 2 Val = 1.4 mM). In general the kinetics were typical of ping‐pong bi‐bi mechanisms in which A 2 Val is the second substrate (intermediate) and O 2 Val is an alternative first substrate. There is no compelling evidence that O 2 Val accepts an amino group at its C5 position resulting in the direct formation of ALev, or the reverse involving the apparent formation of O 2 Val from ALev. These results are consistent with the hypothesis that the mechanism of GSA aminotransferase mimics that of other aminotransferases and that A 2 Val is the intermediate.