Γ-aminobutyric Acid Transaminase Research Articles

Mechanisms of γ-hydroxybutyric acid production during the early postmortem period

Although postmortem production of γ-hydroxybutyric acid (GHB) has been confirmed, its production pathways and substrates have not been sufficiently clarified. To confirm that the residual enzymes are involved in GHB production during the early postmortem period, endogenous GHB concentrations in the postmortem blood and liver samples obtained from charred cadavers were compared with those from noncharred cadavers within 50 h after death. The endogenous GHB concentrations in blood and the livers of charred cadavers were significantly lower than those of noncharred cadavers, showing that heat denaturation of enzyme proteins had prevented the postmortem GHB production. In addition, in vitro experiments with rabbit liver homogenates were carried out for inhibition of postmortem GHB production by specific enzyme inhibitors; N-formylglycine (succinic semialdehyde dehydrogenase inhibitor), valproic acid [γ-aminobutyric acid (GABA) aminotransferase inhibitor], pyrazole (alcohol dehydrogenase inhibitor), or N,N-diethyldithiocarbamic acid (aldehyde dehydrogenase inhibitor) was added and incubated, and the inhibition of GHB production was then measured. The results showed that the inhibition ratios by N-formylglycine, valproic acid, and pyrazole were 74.0%, 10.4%, and 17.7%, respectively. This suggests that the contribution ratios of the GHB-producing pathways originating from succinic acid, GABA/putrescine, and 1,4-butanediol are about 70%, 10%, and 20%, respectively.

Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA Binding

Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.

Synthesis and evaluation of novel aromatic substrates and competitive inhibitors of GABA aminotransferase.

The design, synthesis, and evaluation of novel gamma-aminobutyric acid aminotransferase (GABA-AT) inhibitors and inactivators can lead to the discovery of new GABA-related therapeutics. To this end, a series of aromatic amino acid compounds was synthesized to aid in the design of new inhibitors and inactivators of GABA-AT. All compounds were tested as competitive inhibitors of GABA-AT. The amino acids with benzylic amines were also tested as substrates for GABA-AT. It was found that these compounds were all poor competitive inhibitors of GABA-AT, but some were substrates of the enzyme, suggesting their utility as scaffolds for potential GABA-AT mechanism-based inactivators. Computer modeling was used to rationalize the substrate activity of the various compounds.

Sodium valproate at therapeutic concentrations changes Ca 2+response accompanied with its weak inhibition of protein kinase C in human astrocytoma cells

Sodium valproate (VPA) has been used clinically for treatment of not only epilepsy but also mood disorder. Although VPA is effective for treatment of epilepsy via inhibition of γ-aminobutyric acid transaminase, it remains unknown why VPA is effective for the treatment of mood disorder. The authors examined the effect of VPA at therapeutic concentrations (300 and 600 μM) on the elevation of intracellular free calcium concentration ([Ca 2+] i) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Treatment of the cells with 300 and 600 μM VPA for 2 min did not change the carbachol-induced [Ca 2+] i elevation. Treatment with 300 and 600 μM VPA for 48 h, however, reduced the elevation. Since we have shown that Li + reduced carbachol-induced [Ca 2+] i elevation in protein kinase C (PKC)-downregulated 1321N1 cells [Kurita, M., Mashiko, H., Rai, M., Kumasaka, T., Kouno, S., Niwa, S., Nakahata, N., 2002. Lithium chloride at a therapeutic concentration reduces Ca 2+response in protein kinase C down-regulated human astrocytoma cells, Eur. J. Pharmacol. 442, 17–22.], the activity of PKC was examined. Treatment with VPA at the same concentrations for 24 or 48 h weakly reduced protein kinase C activity in membrane and cytosol fractions from the cells. On the other hand, the treatment of the cells with 600 μM VPA for 24 or 48 h slightly increased the B max value, but not the K d value, in the binding of [ 3H]quinuclidinyl benzylate, a muscarinic receptor ligand, to the membranes, suggesting that the number or affinity of muscarinic receptor did not decrease after VPA treatment. These results indicate that VPA at therapeutic concentrations slightly decreases the PKC activity and inhibits muscarinic receptor-mediated [Ca 2+] i elevation probably through change in the intracellular signaling pathway. VPA-induced reduction of PKC activity and [Ca 2+] i elevation may play a role in the treatment of mood disorder.

Fluorinated Conformationally Restricted γ-Aminobutyric Acid Aminotransferase Inhibitors

On the basis of the structures of several potent inhibitor molecules for gamma-aminobutryric acid aminotransferase (GABA-AT) that were previously reported, six modified fluorine-containing conformationally restricted analogues were designed, synthesized, and tested as GABA-AT inhibitors. The syntheses of all six molecules followed from a readily synthesized ketone intermediate. Three of the molecules were found to be irreversible inhibitors of GABA-AT with comparable or larger k(inact)/K(I) values than that of vigabatrin, a clinically used antiepilepsy drug, and the other three were reversible inhibitors. A possible mechanism for inactivation by one of the inactivators is proposed.

The Sleep-Enhancing Effect of Valerian Inhalation and Sleep-Shortening Effect of Lemon Inhalation

We examined the effects of odorant inhalation on the sleep-wake states in rats. Odorants used in the experiment were clove, jasmine, lavender, lemon, peppermint, pine, rose, sandalwood, valerian, and ylang-ylang. Valerian and rose inhalation significantly prolonged the pentobarbital-induced sleeping time, whereas lemon inhalation significantly shortened it. The effect of valerian inhalation was markedly noticeable. In the anosmic rats, a significant effect of odorants on the pentobarbital sleep time was not seen. Electroencephalographic studies on natural sleep revealed that rose inhalation did not exert any significant effect on sleep, but a significant shortening in sleep latency and a significant prolonging in total sleep time were observed with valerian inhalation, whereas a significant prolonging in sleep latency was observed with lemon inhalation. Such effects of valerian and lemon inhalation were not admitted in anosmic rats. gamma-Aminobutyric acid (GABA) transaminase assay indicates that valerian inhalation decreases the activity of the enzyme and enhances GABA activity. Although valerian has been reported to exert a good effect for sleep as a medicine for internal use, the present study is the first medical report suggesting that the inhalation of valerian may enhance the sleep. On the other hand, the present results may suggest the possibility that lemon inhalation may cause a worsening of insomnia symptoms.

Syntheses and evaluation of fluorinated conformationally restricted analogues of GABA as potential inhibitors of GABA aminotransferase

Inhibition of γ-aminobutyric acid aminotransferase (GABA-AT) could raise the concentration of GABA, an inhibitory neurotransmitter in the human brain, and could have therapeutic applications for a variety of neurological diseases including epilepsy. Four fluorine-containing analogues of GABA with conformations restricted by a cyclohexane ring system were designed and synthesized, but unlike some of their five-membered ring counterparts, minimal inhibition of GABA-AT was observed. It is likely that the rigid chair conformation of these compounds cannot be accommodated well in the enzyme’s active site.

New substrates and inhibitors of γ-aminobutyric acid aminotransferase containing bioisosteres of the carboxylic acid group: Design, synthesis, and biological activity

A series of potential substrates of γ-aminobutyric acid aminotransferase (GABA-AT) with lipophilic bioisosteres of the carboxylic acid group ( 2– 7) were synthesized and tested. Most of the synthesized compounds showed substrate activities with GABA-AT; 1 H-tetrazole-5-propanamine ( 6) was the best of those tested. The potential time-dependent inhibitor of GABA-AT, 1 H-tetrazole-5-(α-vinyl-propanamine) ( 8), was designed based on the structures of 6 and the antiepilepsy drug vigabatrin (4-aminohex-5-enoic acid, 1). The synthesized compound 8 showed time-dependent inhibition of GABA-AT, but its potency is lower than that of vigabatrin. Methylation of the tetrazole group in 8 resulted in loss of time-dependent activity, suggesting that the tetrazole ring, the carboxylate bioisostere, exists in its deprotonated form in the enzyme active site.

Design and biological evaluation of phenyl-substituted analogs of β-phenylethylidenehydrazine

β-Phenylethylidenehydrazine (PEH) has been demonstrated previously to be an inhibitor of γ-aminobutyric acid transaminase (GABA-T) and to cause a marked increase in rat brain levels of GABA, a major neurotransmitter. A group of PEH analogs, possessing a variety of substituents (Me, OMe, Cl, F, and CF3) at the 2-, 3-, and 4-positions of the phenyl ring, were synthesized for evaluation as inhibitors of GABA-T. The details of the synthesis and chemical characterization of the analogs are described. Preliminary in vitro screening for GABA-T inhibition showed that all the analogs possessed activity against this enzyme, although substitution of CF3 at the 2- and 4-positions caused reduced activity. One of the drugs, 4-fluoro-β-phenylethylidenehydrazine, was investigated further ex vivo, where it was shown to inhibit GABA-T, elevate brain levels of GABA, and decrease levels of glutamine, similar to the profile observed previously for PEH.

Effects of sodium nitroprusside, a nitric oxide donor, on γ-aminobutyric acid concentration in the brain and on picrotoxin-induced convulsions in combination with phenobarbitone in rats

The concentrations of nitric oxide (NO), the neuronal messenger molecule, and γ-aminobutyric acid (GABA), the inhibitory neurotransmitter, and the activity of γ-aminobutyric acid transaminase (GABA-T), the enzyme involved in the degradation of GABA, were measured in the brain of rats treated with graded doses (1.25, 2.5, 5.0 mg/kg) of sodium nitroprusside (SNP), the donor of NO. The effect of SNP was tested alone and in combination with phenobarbitone (PB), the GABA potentiating antiepileptic drug, against picrotoxin (PCT) (5 mg/kg)-induced convulsions in rats. The results of these studies showed that NO released from SNP (2.5 mg/kg) had a potential to inhibit GABA-T activity resulting in an increase in the concentration of GABA in the brain. Thus, SNP (2.5 mg/kg) was able to inhibit PCT-induced convulsions and was able to produce an additive anticonvulsant action with PB. However, a much greater increase in the concentration of NO by 5.0 mg/kg of SNP did not change the activity of GABA-T and the concentration of GABA, and promoted the convulsant action of PCT. These results suggest that a moderate increase in the concentration of NO following the administration of its donor SNP (2.5 mg/kg) results in an enhancement of the concentration of GABA in the brain and in an inhibition of PCT-induced convulsions independently and additively with PB and that a marked increase in NO concentration after the administration of a larger dose of SNP (5.0 mg/kg) results in proconvulsant action.

Mechanistic Crystallography. Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1R,3S,4S)-3-Amino-4-fluorocyclopentane-1-carboxylic Acid As Elucidated by Crystallography

(1R,3S,4S)-3-Amino-4-fluorocyclopentane-1-carboxylic acid (7) was previously shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) [Qiu, J. and Silverman, R. B. (2000) J. Med. Chem. 43, 706-720]. Two mechanisms were considered as reasonable possibilities, a Michael addition mechanism and an enamine mechanism. On the basis of a variety of chemical studies, including tedious radiolabeling experiments, it was concluded that inactivation by 7 proceeds by a Michael addition mechanism. Here, a crystal structure of 7 bound to pig liver GABA-AT is reported, which clearly demonstrates that the adduct formed is derived from an enamine mechanism. This represents another example of how crystallography is an important tool for elucidation of inactivation mechanisms.

Conformationally-restricted vigabatrin analogs as irreversible and reversible inhibitors of gamma-aminobutyric acid aminotransferase.

Compounds that inhibit gamma-aminobutyric acid aminotransferase exhibit anticonvulsant activity; vigabatrin is a known irreversible inhibitor of this enzyme and anticonvulsant drug. Conformationally-restricted, five-membered- and six-membered-ring vigabatrin analogs were synthesized and tested as inhibitors of gamma-aminobutyric acid aminotransferase. Two monofluorinated compounds, 4 and 5, are time-dependent inhibitors of the enzyme, and their potencies are comparable to that of vigabatrin. Compounds 6 and 7 are weak reversible inhibitors.

Synthesis of Cyclopropane Isosteres of the Antiepilepsy Drug Vigabatrin and Evaluation of their Inhibition of GABA Aminotransferase

The antiepilepsy drug vigabatrin (1; 4-aminohex-5-enoic acid; γ-vinyl GABA) is a mechanism-based inactivator of the pyridoxal 5'-phosphate (PLP)-dependent enzyme γ-aminobutyric acid aminotransferase (GABA-AT). Inactivation has been shown to proceed by two divergent mechanisms (Nanavati, S. M. and Silverman, R. B. (1991) J. Am. Chem. Soc. 113, 9341–9349), a Michael addition pathway (Scheme 2, pathway a) and an enamine pathway (Scheme 2, pathway b). Analogs of vigabatrin with a cyclopropyl or cyanocyclopropyl functionality in place of the vinyl group (2–5) were synthesized as potential inactivators of GABA-AT that can inactivate the enzyme only through a Michael addition pathway, but they were found to be only weak inhibitors of the enzyme.

Age-related changes of γ-aminobutyric acid transaminase immunoreactivity in the hippocampus and dentate gyrus of the Mongolian gerbil

We investigated the age-related changes of γ-aminobutyric acid transaminase (GABA-T, a GABA degradation enzyme) in the hippocampus and dentate gyrus of the gerbil at postnatal month 1 (PM 1), PM 3, PM 6, PM 12, and PM 24. Age-related changes of GABA-T immunoreactivity were distinct in the hippocampal CA1 region and in the dentate gyrus. GABA-T immunoreactivity was weak at PM 1, but at PM 3, it had increased significantly, and then increased further. Between PM 6 and PM 12, strong GABA-T immunoreactivity was found in nonpyramidal cells (GABAergic) in the stratum pyramidale of the CA1 region, and at PM 6, strong GABA-T immunoreactivity was found in neurons of the dentate gyrus subgranular zone. At PM 24, CA1 pyramidal cells showed strong GABA-T immunoreactivity. Western blot analysis showed a pattern of GABA-T expression similar to that shown by immunohistochemistry at various ages. In conclusion, our results suggest that the age-related changes of GABA-T provide important information about the aged brain with GABA dysfunction.

Structures of γ-Aminobutyric Acid (GABA) Aminotransferase, a Pyridoxal 5′-Phosphate, and [2Fe-2S] Cluster-containing Enzyme, Complexed with γ-Ethynyl-GABA and with the Antiepilepsy Drug Vigabatrin

Gamma-aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, gamma-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-A resolution) and in complex with vigabatrin as well as with the close analogue gamma-ethynyl-GABA (to 2.3 and 2.8 A, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5'-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.

Valproate prevents the induction, but not the expression of morphine sensitization in mice

Repetitive exposure to opioids elicits sensitization to its locomotor stimulating effects. Several lines of evidence have shown that the central GABAergic system is involved in behavioral sensitization induced by morphine. Valproate, a clinically widely used anticonvulsant and mood stabilizer, can mainly inhibit γ-aminobutyric acid (GABA) transaminase and activate glutamic acid decarboxylase, which result in decrease in the degradation and increase in the synthesis of GABA, and then the elevation of extracellular GABA in the central nervous system. However, the effects of valproate on behavioral sensitization to morphine have not been documented. Herein, we investigated the effects of valproate on the induction and the expression of behavioral sensitization to morphine. Mice treated daily for 7 days with 10 mg/kg morphine and challenged with the same dose after 7 days of washout showed increased locomotor activity. Co-administration of valproate (37.5, 75, 150 mg/kg, intraperitoneal (i.p.)), at doses that did not affect the spontaneous activity, 30 min prior to morphine dose-dependently inhibited the induction of morphine sensitization. However, neither single nor multiple administration (37.5, 75, 150 mg/kg×7 injections) of valproate had any effect on the expression of morphine sensitization once it developed. Our results indicated that GABA plays an important role in the induction, but not in the expression of morphine sensitization in mice.

Design, synthesis, and biological activity of a difluoro-substituted, conformationally rigid vigabatrin analogue as a potent gamma-aminobutyric acid aminotransferase inhibitor.

Previously it was found that a conformationally rigid analogue (2) of the epilepsy drug vigabatrin (1) did not inactivate gamma-aminobutyric acid aminotransferase (GABA-AT). A cyclic compound with an exocyclic double bond (6) was synthesized and was found to inactivate GABA-AT, but only in the absence of 2-mercaptoethanol. The corresponding difluoro-substituted analogue (14) was synthesized and was shown to be a very potent time-dependent inhibitor, even in the presence of 2-mercaptoethanol.

Inactivation of γ-aminobutyric acid aminotransferase by ( S)-4-amino-4,5-dihydro-2-furancarboxylic acid does not proceed by the expected aromatization mechanism

Inactivation of pyridoxal 5′-phosphate (PLP)-dependent γ-aminobutryic acid aminotransferase by ( S)-4-amino-4,5-dihydro-2-furancarboxylic acid (SADFA) gives pyridoxamine 5′-phosphate, not the expected SADFA-PLP aromatization product. Inactivation appears to proceed by a Michael addition/hydrolysis mechanism instead.

Kinetic Studies on the Inhibition of GABA-T by γ-Vinyl GABA and Taurine

γ-Aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of γ-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: γ-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by γ-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for γ-vinyl GABA and taurine were found to be 26±3 mM and 68±7 mM respectively. The transamination process of α-ketoglutarate was not affected by the presence of γ-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when α-ketoglutarate was the substrate. The inhibition dissociation constant (Kii) for this system was found to be 96±10 mM. The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79±0.11 mM, and 0.47±0.10 mM for GABA and α-ketoglutarate respectively.

GABA Guides the Pollen Tube

Plant fertilization involves the delivery of the sperm from the pollen, which lands at the top of the flower, to the ovule by the directed growth of the pollen tube. Palanivelu et al. determined that POP2 , a gene implicated in pollination by genetic analysis, encodes a γ-aminobutyric acid (GABA) transaminase. In Arabidopsis , POP2 mutant plants have a 100-fold increase in the concentration of GABA in flowers compared with wild-type flowers. Analysis of POP2 mutants showed that the severity of the seed phenotype correlated with the GABA concentration. Analysis of wild-type flowers showed that GABA exists in a gradient--lowest at the peripheral tissues, the stigma, and highest in the cells surrounding the micropyle (integument cells). The micropyle is the place where the pollen tube enters the ovule. Fertilization of wild-type or POP2 -mutant females by wild-type or mutant pollen showed that only one, either the male or female, needed to be wild type in order for proper pollen tube guidance and seed production. If mutant flowers were self-pollinated, the pollen tubes did not navigate properly, and seed generation was poor. In POP2 -mutant females, a gradient of GABA still persists at the micropyle; however, GABA is more abundant in other parts of the flower. The authors suggest that, when pollinated by wild-type pollen, the POP2 GABA transaminase in the pollen tube can enhance the gradient and allow fertilization, whereas POP2 -mutant pollen cannot create an adequate gradient. Surprisingly, in vitro or in vivo pollen tubes do not grow toward an exogenous GABA source. In vitro, pollen tubes do elongate in low concentrations of GABA, but do not elongate in high concentrations of GABA. The results suggest that, in plants, a combination of nonuniform GABA synthesis and controlled degradation serve to create a gradient that helps guide the pollen tube to the ovule. R. Palanivelu, L. Brass, A. F. Edlund, D. Preuss, Pollen tube growth and guidance is regulated by POP2 , an Arabidopsis gene that controls GABA levels. Cell 114 , 47-59 (2003). [Online Journal]