CHARACTERIZATION OF THE DIFFERENT SPECTRAL FORMS OF GLUTAMATE 1-SEMIALDEHYDE AMINOTRANSFERASE BY MASS-SPECTROMETRY

Glutamate 1-semialdehyde aminotransferase produces delta-aminolevulina te for the synthesis of chlorophyll, heme, and other tetrapyrrole pigm ents. The native enzyme from Synechococcus is pale yellow and has abso rption maxima at 338 and 418 nm from vitamin B-6. Yellow, colorless, a nd pink forms of the protein are obtained by treatment with 4,5-dioxov alerate, 4,5-diaminovalerate, and acetylenic GABA, respectively. Compa red to the native enzyme, the 418 nm absorption maximum in the yellow enzyme is enhanced and the 338 nm maximum reduced while the colorless enzyme has a heightened maximum at 338 nm and a barely detectable peak at 418 nm. The pink enzyme has an absorption maximum at 560 nm. When the native and colorless enzymes are repeatedly diluted in 0.5 M Na2HP O4, pH 7.0, and reconcentrated, pyridoxamine 5'-phosphate is released and the 338 nm maximum lost. Thus the 338 nm absorption maximum is ass ociated with noncovalently bound pyridoxamine 5'-phosphate. NaBH4 redu ction proved that the absorbance at 418 nm is from pyridoxal 5'-phosph ate cofactor bound by a Schiff base to the protein. When the native, c olorless, and yellow enzymes were subjected to electrospray ionization mass spectrometry, the Bg cofactor dissociated from the protein and g ave a molecular weight of 46 401-46 418. Acetylenic GABA and NaBH4 wer e used for protein modification, and they reacted with the native and yellow enzymes but had no effect on the colorless enzyme. Pyridoxal 5' -phosphate bound covalently to the protein after NaBH4 reduction. Acet ylenic GABA attached covalently to the enzyme produced an additional m ass peak, 123-126 mass units higher, in the electrospray ionization sp ectrum. Tryptic peptide analysis showed no disulfide bonds in glutamat e 1-semialdehyde aminotransferase and that Lys(276) is the binding sit e for both acetylenic GABA and pyridoxal 5'-phosphate. A likely mechan ism is suggested for covalent binding of acetylenic GABA to Lys(276).