A Gaba Synthesis

GAD consists almost entirely of homodimers of two distinct polypeptides—GAD65 (with Mr of 65,000)

Glutamate Synthesis The Brain

Figure 1 Schematic of GABA-related proteins and their locations within the cell. GAD65 or GAD67 synthesizes GABA from glutamate. GABA can be packaged into vesicles for release (vesicular GABA transporter not shown) at the synapse, where it binds to GABA receptors. GABA receptors are located on the presynaptic and postsynaptic neuron and can be found outside of the synapse. The plasma membrane GABA transporter (GAT) takes up unbound GABA from the synapse. GABA can also exit the cell via GAT. The GABA shunt is depicted within the mitochondria. Also shown are two sources of glutamate: a-Ketoglutarate can be converted to glutamate by GABA-T, and glutamine, provided by astrocytes, can be converted to glutamate by phosphate-activated glutaminase (PAG). GABAR, GABA receptor; GAD, glutamic acid decarboxylase; GABA-T, GABA-transaminase; a-kg, a-ketoglutarate; Succ, succinate; SSA, succinic semialdehyde; SSADH, succinic semialdehyde dehydrogenase; Gln, glutamine [from D. L. Martin and A. J. Tobin, Mechanisms controlling GABA synthesis and degradation in the brain. In GABA in the Nervous System: The View at Fifty Years (D. L. Martin and R. W. Olsen, Eds.). Lippincott, Williams & Wilkins, Philadelphia, 2000].

Figure 1 Schematic of GABA-related proteins and their locations within the cell. GAD65 or GAD67 synthesizes GABA from glutamate. GABA can be packaged into vesicles for release (vesicular GABA transporter not shown) at the synapse, where it binds to GABA receptors. GABA receptors are located on the presynaptic and postsynaptic neuron and can be found outside of the synapse. The plasma membrane GABA transporter (GAT) takes up unbound GABA from the synapse. GABA can also exit the cell via GAT. The GABA shunt is depicted within the mitochondria. Also shown are two sources of glutamate: a-Ketoglutarate can be converted to glutamate by GABA-T, and glutamine, provided by astrocytes, can be converted to glutamate by phosphate-activated glutaminase (PAG). GABAR, GABA receptor; GAD, glutamic acid decarboxylase; GABA-T, GABA-transaminase; a-kg, a-ketoglutarate; Succ, succinate; SSA, succinic semialdehyde; SSADH, succinic semialdehyde dehydrogenase; Gln, glutamine [from D. L. Martin and A. J. Tobin, Mechanisms controlling GABA synthesis and degradation in the brain. In GABA in the Nervous System: The View at Fifty Years (D. L. Martin and R. W. Olsen, Eds.). Lippincott, Williams & Wilkins, Philadelphia, 2000].

and GAD67 (with Mr of 67,000). The two GADs account for essentially all GAD activity in tissue extracts. Although other purified proteins reportedly have GAD activity, none is known to contribute substantially to GABA synthesis in vivo.

The two GAD polypeptides are the products of distinct genes and differ in sequence by about 35%. The two genes have identical exon-intron organizations, suggesting that they share a relatively recent common ancestor. The GAD65s of humans, mice, and rats are 97% identical, as are the GAD67s of humans, rats, and cats. Clearly, the two GADs have been under strong selective pressure during the 150-200 million years since the beginning of mammalian radiation.

Almost every neuron that produces one form of GAD also produces the other, suggesting that the two genes share some transcriptional regulatory mechanisms. During the development of specific brain structures, GAD67 usually appears earlier than GAD65. In the hippocampus and the spinal cord, however, GAD65 appears earlier in development than GAD67. The ratio of GAD65 to GAD67 mRNA increases dramatically during synapse formation, both in the striatum and in the cerebellum.

Glutamate Dehydrogenase Mechanism
Figure 2 GABA is synthesized from the decarboxylation of glutamate. GABA is degraded to succinic semialdehyde and succinate by GABA-T and succinic semialdehyde dehydrogenase (SSADH). GABA-T can also convert a-ketoglutarate to glutamate, completing the first step of the GABA shunt.

The GAD67 gene produces two alternatively spliced transcripts during early development. The two embryonic forms of GAD are GAD25 and GAD44, but only GAD44 is enzymatically active. GAD25 is not enzymatically active because it lacks the PLP binding site. As the embryo develops, the shorter transcripts are replaced by the mature GAD67. GAD65 has no identified alternatively spliced transcripts.

GAD65 and GAD67 differ in their enzymatic characteristics. GAD67 exists mainly in the active, holoen-zyme form (bound to PLP) and is less sensitive to small changes in neuronal GABA concentration than is GAD65. Most GAD65 in the brain is in the apoenzyme form (inactive, not bound to PLP), and the GAD activity of brain extracts increases two- or threefold upon the addition of PLP. The loss of PLP from GAD is not a simple dissociation but a catalytic misstep that results in the formation of succinic semialdehyde and pyridoxamine phosphate (rather than GABA and PLP). This reaction is illustrated in Fig. 3. Pyridox-amine phosphate dissociates from GAD, generating an apoenzyme that lacks enzymatic activity until it again combines with PLP to reform holoGAD65.

The interconversion of apoGAD and holoGAD is sensitive to ATP, phosphate (Pi), and GABA levels.

These influences may regulate GABA production in response to altered neuronal activity within GABA neurons. ATP favors the formation of apoGAD and inhibits the formation of holoGAD. Pi enhances the formation of holoGAD. GABA also favors apoGAD formation in the absence of PLP because the GABA-forming steps are readily reversible. Consequently, GAD65 is sensitive to small changes in neuronal GABA concentration. GAD has a Km for glutamate of approximately 0.45 mM and a Km for GABA of approximately 16 mM. Therefore, given similar concentrations of glutamate and GABA, glutamate is converted to GABA much faster than the reverse reaction.

GAD65 and GAD67 also differ in their sub-cellular locations. Both are present in cell bodies and axon terminals, but GAD65 is usually more concentrated in axon terminals, whereas GAD67 is more concentrated in cell bodies. GAD65 associates with vesicle membranes both in neurons and in pancreatic cells, and it is characterized by punctate immuno-staining in mature neurons. GAD67 generally shows no such association with vesicles, and it is diffuse throughout the cell when examined by immuno-staining.

Figure 3 Interconversion of the GAD apoenzyme and holoenzyme. GAD can be converted from its active holoenzyme form, bound to pyridoxal phosphate (PLP), into its inactive apoenzyme form by a catalytic misstep that results in the formation of pyridoxal monophosphate (PMP) and succinic semialdehyde (SSA). GAD must reassociate with PLP to be active [adapted from Brain Glutamate Decarboxylase. In Neurotransmitter enzymes (Boulton, Baker, and Yu, Eds.) Humana Press, Clifton, NJ].

Figure 3 Interconversion of the GAD apoenzyme and holoenzyme. GAD can be converted from its active holoenzyme form, bound to pyridoxal phosphate (PLP), into its inactive apoenzyme form by a catalytic misstep that results in the formation of pyridoxal monophosphate (PMP) and succinic semialdehyde (SSA). GAD must reassociate with PLP to be active [adapted from Brain Glutamate Decarboxylase. In Neurotransmitter enzymes (Boulton, Baker, and Yu, Eds.) Humana Press, Clifton, NJ].

GAD65 undergoes at least two types of reversible posttranslational modifications—palmitoylation and phosphorylation. These modifications can anchor GAD65 to internal membranes, even in cells not specialized for exocytosis, but they are not required for membrane association. Palmitoylation and phosphorylation are limited to a distinct GAD65 polypep-tide whose electrophoretic mobility is slightly less than that of GAD65, but the structural basis for this difference in mobility is not well understood.

Although most GAD molecules are homodimers, GAD65 and GAD67 also form heterodimers. The association of some GAD67 with synaptic terminals and with the Golgi complex in genetically modified cells appears to depend on its association with membrane-targeted GAD65. The presence of GAD67 in a restricted subset of synaptic boutons, observed in the mouse hippocampus, may reflect the distribution of GAD65-GAD67 heterodimers.

duces one NADH and one GTP. The GABA shunt therefore produces approximately 8% less total energy compared to the TCA cycle, although it provides 1020% of the TCA cycle activity in most brain regions.

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