Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis

Glutamate decarboxylase (GAD) exists as two isoforms, GAD(65) and GAD(67) . GAD activity is regulated by a cycle of activation and inactivation determined by the binding and release of its co-factor, pyridoxal 5'-phosphate. Holoenzyme (GAD with bound co-factor) decarboxylates glutamate to form GABA, but it also catalyzes a slower transamination reaction that produces inactive apoGAD (without bound co-factor). Apoenzyme can reassociate with pyridoxal phosphate to form holoGAD, thus completing the cycle. Within cells, GAD(65) is largely apoenzyme (similar to93%) while GAD(67) is mainly holoenzyme (similar to72%). We found striking kinetic differences between the GAD isoforms that appear to account for this difference in co-factor saturation. The glutamate dependent conversion of holoGAD(65) to apoGAD was about 15 times faster than that of holoGAD(67) at saturating glutamate. Aspartate and GABA also converted holoGAD(65) to apoGAD at higher rates than they did holoGAD(67) . Nucleoside triphosphates (such as ATP) are known to affect the activation reactions of the cycle. ATP slowed the activation of GAD(65) and markedly reduced its steady-state activity, but had little affect on the activation of GAD(67) or its steady-state activity. Inorganic phosphate opposed the effect of ATP; it increased the rate of apoGAD(65) activation but had little effect on apoGAD(67) activation. We conclude that the apo-/holoenzyme cycle of inactivation and reactivation is more important in regulating the activity of GAD(65) than of GAD(67.)