Spectral kinetics of glutamate-1-semialdehyde aminomutase of Synechococcus.

Abstract

Purified Synechococcus glutamate-1-semialdehyde aminotransferase (GSA-AT; EC 5.4.3.8) has absorption maxima characteristic of vitamin B6-containing enzymes and can be converted to the pyridoxamine 5'-phosphate or pyridoxal 5'-phosphate form by reaction with diaminovalerate or dioxovalerate, respectively, suggesting that these two analogues are intermediates in the conversion of glutamate 1-semialdehyde (GSA) to 5-aminolevulinate (ALA). Values for Km and kmax were calculated for GSA, diaminovalerate, ALA, and gabaculine from absorption change rates during conversion of one coenzyme form of GSA-AT to the other, upon addition of one of these compounds. The substrate specificity (kmax/Km) of diaminovalerate is about 3 orders of magnitude larger than that of dioxovalerate, making the latter an unlikely intermediate in the enzymic conversion of GSA to ALA. GSA reacts with both coenzyme forms, whereas ALA only reacts with the pyridoxamine 5'-phosphate form of the enzyme. However, ALA does form a complex with the pyridoxal 5'-phosphate form of GSA-AT and inhibits reactions between gabaculine and GSA-AT. This relatively stable complex (Ki = 8 M) may have significance in enzyme inhibition. Both L and D enantiomers of GSA react with GSA-AT. Spectral changes observed upon addition of DL-GSA are apparently due to reaction with the less reactive D-isomer. L-GSA is converted to ALA prior to major spectral changes induced by the racemic mixture.