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 microcalo rimetry. The enzyme was prepared from Sf9 insect cells infected with a recombinant baculovirus containing the entire GAD65 coding region, Th e UV/visible absorption spectrum of purified holoenzyme (holoGAD) exhi bits two peaks in the range of 300-450 nm, which are due to the format ion of a Schiff base when pyridoxal phosphate (pyridoxal-P) binds to G AD. Fluorescence emission intensity (excited at 295 or 280 nm) was sub stantially enhanced when pyridoxal-P was removed from holoGAD and quen ched when pyridoxal-P was added to the apoenzyme (apoGAD). These obser vations implied that a significant enzyme conformational change occurs during the formation of holoGAD. Circular dichroism provided addition al evidence for a conformational change, as the ellipticity of both ne gative (202-242 nm) and positive (188-202 nm) bands decreased when pyr idoxal-P was removed from holoGAD. Secondary structure determination e stimated that holoGAD contains a higher content of alpha-helix (34% ve rsus 24%) and a lower content of beta-sheet (18% versus 30%) than apoG AD. Differential scanning microcalorimetry indicated that holoGAD exhi bits a much larger enthalpy and a 3 degrees C higher temperature of th ermal unfolding than apoGAD, suggesting that holoGAD has a much tighte r conformation and greater stability than apoGAD. A model describing t he interaction of pyridoxal-P with GAD is presented, which proposes th at an intermediate complex involving ionic interaction between the pho sphate group of pyridoxal-p and the positive, charged residues in the active site of GAD maintains the pyridoxal-P molecule in an appropriat e position irl the active center. Simultaneously, this complex formati on is accompanied by a moderate enzyme conformational change, providin g a favorable configuration that enables the epsilon-amino of the acti ve-site lysine to react with the aldehyde group of pyridoxal-P, The fo rmation of active holoGAD involves a large enzyme conformational chang e, which leads to increased stability. (C) 1998 Academic Press.