Mechanism of glutamate semialdehyde aminotransferase. Roles of diamino- and dioxo-intermediates in the synthesis of aminolevulinate.
Glutamate semialdehyde aminotransferase was purified to homogeneity from pea leaves. The enzyme has an absorption spectrum with maxima at 345 and 416 nm. These chromophores were attributed to pyridoxamine phosphate and to pyridoxal phosphate bound as an aldimine respectively. Treatment of the enzyme with increasing concentrations of diaminovalerate produced a rapid fall in the 416 nm chromophore and a simultaneous increase in the 345 nm chromophore in a manner that indicated a stoichiometric reaction between 4,5-diaminovalerate and the pyridoxaldimine form of the enzyme. Treatment with 4,5-dioxovalerate produced the reverse reaction. The enzyme catalyzed the formation of aminolevulinate when dioxovalerate and diaminovalerate were present together and the maximal rate was 40% of that observed when glutamate semialdehyde itself was used as substrate. Conversion of the enzyme from its pyridoxaldimine to pyridoxamine form produced a proportional increase in activity towards glutamate semialdehyde, whereas reduction of the pyridoxaldimine form with sodium borohydride produced no change in this catalytic activity. It was concluded, therefore, that only the pyridoxamine form of the enzyme is active in catalyzing conversion of glutamate semialdehyde to aminolevulinate and that the catalytic mechanism includes enzyme-bound diaminovalerate as a central intermediate.
Pugh
C E
CE
Department of Biochemistry, University of Wales College of Cardiff, United Kingdom.
Harwood
J L
JL
John
R A
RA
D000622:Aminolevulinic Acid / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism; D019751:Intramolecular Transferases; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008968:Molecular Conformation; D015394:Molecular Structure; D010946:Plants, Medicinal; D013053:Spectrophotometry
D005971:Glutamates; C021199:glutamic acid gamma-semialdehyde; D000622:Aminolevulinic Acid; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase