Biosynthesis of delta-aminolevulinate in greening barley leaves. IX. Structure of the substrate, mode of gabaculine inhibition, and the catalytic mechanism of glutamate 1-semialdehyde aminotransferase.
Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by 1H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 min. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate 1-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the absence of glutamate 1-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.
Hoober
J K
JK
Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140.
Kahn
A
A
Ash
D E
DE
Gough
S
S
Kannangara
C G
CG
D000622:Aminolevulinic Acid / Q000096:biosynthesis*; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology*; D002523:Edible Grain / Q000378:metabolism*; D001467:Hordeum / Q000378:metabolism*; D019751:Intramolecular Transferases; D007982:Levulinic Acids / Q000096:biosynthesis*; D000637:Transaminases / Q000032:analysis* / Q000037:antagonists & inhibitors
D003509:Cyclohexanecarboxylic Acids; D007982:Levulinic Acids; C012987:gabaculine; D000622:Aminolevulinic Acid; D000637:Transaminases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase