Structural Characteristics of Brain Glutamate Decarboxylase in Relation to Its Interaction and Activation

The conformation, stability, cofactor interactions, and activation of a recombinant 65-kDa form of rat brain glutamate decarboxylase (GAD65) were investigated by using UV/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. The enzyme was prepared from Sf9 insect cells infected with a recombinant baculovirus containing the entire GAD65 coding region. The UV/visible absorption spectrum of purified holoenzyme (holoGAD) exhibits two peaks in the range of 300–450 nm, which are due to the formation of a Schiff base when pyridoxal phosphate (pyridoxal-P) binds to GAD. Fluorescence emission intensity (excited at 295 or 280 nm) was substantially enhanced when pyridoxal-P was removed from holoGAD and quenched when pyridoxal-P was added to the apoenzyme (apoGAD). These observations implied that a significant enzyme conformational change occurs during the formation of holoGAD. Circular dichroism provided additional evidence for a conformational change, as the ellipticity of both negative (202–242 nm) and positive (188–202 nm) bands decreased when pyridoxal-P was removed from holoGAD. Secondary structure determination estimated that holoGAD contains a higher content of α-helix (34% versus 24%) and a lower content of β-sheet (18% versus 30%) than apoGAD. Differential scanning microcalorimetry indicated that holoGAD exhibits a much larger enthalpy and a 3°C higher temperature of thermal unfolding than apoGAD, suggesting that holoGAD has a much tighter conformation and greater stability than apoGAD. A model describing the interaction of pyridoxal-P with GAD is presented, which proposes that an intermediate complex involving ionic interaction between the phosphate group of pyridoxal-P and the positive, charged residues in the active site of GAD maintains the pyridoxal-P molecule in an appropriate position in the active center. Simultaneously, this complex formation is accompanied by a moderate enzyme conformational change, providing a favorable configuration that enables the ϵ-amino of the active-site lysine to react with the aldehyde group of pyridoxal-P. The formation of active holoGAD involves a large enzyme conformational change, which leads to increased stability.