Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis.
Glutamate decarboxylase (GAD) exists as two isoforms, GAD65 and GAD67. 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, GAD65 is largely apoenzyme (approximately 93%) while GAD67 is mainly holoenzyme (approximately 72%). 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 holoGAD65 to apoGAD was about 15 times faster than that of holoGAD67 at saturating glutamate. Aspartate and GABA also converted holoGAD65 to apoGAD at higher rates than they did holoGAD67. Nucleoside triphosphates (such as ATP) are known to affect the activation reactions of the cycle. ATP slowed the activation of GAD65 and markedly reduced its steady-state activity, but had little affect on the activation of GAD67 or its steady-state activity. Inorganic phosphate opposed the effect of ATP; it increased the rate of apoGAD65 activation but had little effect on apoGAD67 activation. We conclude that the apo-/holoenzyme cycle of inactivation and reactivation is more important in regulating the activity of GAD65 than of GAD67.
Battaglioli
Gino
G
Wadsworth Center, New York State Department of Health, Albany, New York, USA. battagli@wadsworth.org
Liu
Hongcheng
H
Martin
David L
DL
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D018411:Spodoptera; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis* / Q000494:pharmacology
D001051:Apoenzymes; D020035:Holoenzymes; D007527:Isoenzymes; D000266:Adenylyl Imidodiphosphate; D001224:Aspartic Acid; D018698:Glutamic Acid; D000249:Adenosine Monophosphate; D005680:gamma-Aminobutyric Acid; D000255:Adenosine Triphosphate; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2