Characterization of the different spectral forms of glutamate 1-semialdehyde aminotransferase by mass spectrometry.
Glutamate 1-semialdehyde aminotransferase produces delta-aminolevulinate for the synthesis of chlorophyll, heme, and other tetrapyrrole pigments. The native enzyme from Synechococcus is pale yellow and has absorption maxima at 338 and 418 nm from vitamin B6. Yellow, colorless, and pink forms of the protein are obtained by treatment with 4,5-dioxovalerate, 4,5-diaminovalerate, and acetylenic GABA, respectively. Compared 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 Na2HPO4, pH 7.0, and reconcentrated, pyridoxamine 5'-phosphate is released and the 338 nm maximum lost. Thus the 338 nm absorption maximum is associated with noncovalently bound pyridoxamine 5'-phosphate. NaBH4 reduction proved that the absorbance at 418 nm is from pyridoxal 5'-phosphate cofactor bound by a Schiff base to the protein. When the native, colorless, and yellow enzymes were subjected to electrospray ionization mass spectrometry, the B6 cofactor dissociated from the protein and gave a molecular weight of 46,401-46,418. Acetylenic GABA and NaBH4 were 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. Acetylenic GABA attached covalently to the enzyme produced an additional mass peak, 123-126 mass units higher, in the electrospray ionization spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)
Brody
S
S
Department of Biology, New York University, New York 10003, USA.
Andersen
J S
JS
Kannangara
C G
CG
Meldgaard
M
M
Roepstorff
P
P
von Wettstein
D
D
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D004926:Escherichia coli; D019751:Intramolecular Transferases; D007535:Isomerases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods
D001051:Apoenzymes; D003509:Cyclohexanecarboxylic Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; C012987:gabaculine; D011733:Pyridoxamine; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase; C010627:pyridoxamine phosphate