Glutamate 1-semialdehyde aminotransferase: anomalous enantiomeric reaction and enzyme mechanism.
Glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes near 50% conversion of the racemic mixture of GSA to 5-aminolevulinate (ALA), indicating quantitative use of the L-glutamate-derived natural (S)-enantiomer as substrate. This enzymic reaction has been extensively studied with (R,S)-GSA because it is readily purified in high yields following ozonolysis of racemic 4-vinyl-4-aminobutyric acid. However upon addition of (R,S)-GSA, GSA-aminotransferase is converted to the pyridoxal-P or internal aldimine form (418 nm) and not rapidly cycled back to the original pyridoxamine-P, as predicted by the rate of product (ALA) accumulation. Addition of the putative intermediate, (R,S)-4,5-diaminovalerate (DAVA), eliminates this rapid conversion of the enzyme by (R,S)-GSA to the internal aldimine and stimulates initial rates of ALA synthesis (2-3-fold) and results in corresponding increases in apparent equilibrium concentrations of ALA. These results indicate that DAVA is rate limiting and suggest anomalous reactivity of (R)-GSA. Steady-state and spectral kinetic experiments with individual purified enantiomers confirm anomalous reactivity of (R)-GSA: in the case of (S)-GSA, spectral changes are lesser in amplitude and at least 1 or 2 orders of magnitude more rapid. Only (S)-GSA yielded significant amounts of ALA. Since (R)-GSA is an apparent substrate in the first half-reaction, the resulting (R)-DAVA is either inactive or a poor substrate in the second half-reaction.