Some molecular properties of glutamate decarboxylase from rice germ

Glutamate decarboxylase (EC 4.1.1.15, GAD) is a pyridoxal 5′-phosphate (PLP) dependent enzyme, which catalyses the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid (GABA). GAD was purified 186-fold from rice germ. Ultraviolet–visible spectra showed that the rice germ holoGAD presented a weak peak at 420 nm, but the inactivated apoGAD did not. The holoGAD also exhibited a strong peak at 308 nm and a weak peak at 336 nm in its fluorescence emission spectrum. The apoGAD led to a 20% increase in the fluorescence emission at 308 nm. The contents of the secondary structure elements of the holoGAD and apoGAD were estimated from the values of the mean residue ellipticity based on the CD spectra. The holoGAD had a greater β-sheet content than the apoGAD (39% versus 27%), whereas both had a similar α-helix content (13% versus 14%). These findings confirmed that a slight conformational change had occurred when PLP bound to the apoGAD to form the holoGAD. Chemical modifications of the GAD by some selected reagents indicated that histidine residue(s) might be involved in the enzymatic functions, but were not essential for the enzyme activity. The study also suggested there was one arginine residue in the GAD active site, and most likely at the substrate-binding site.