0020-711X/90$3.00+ 0.00 Copyright 0 1990Perpmon Press plc
fnr. J. Biochem.Vol. 22, No. 11, pp. 1235-1241,1990 Printed in Great Britain. All rights reserved
MINIREVIEW ACTION
OF INHIBITORS ON BRAIN GLUTAMATE DECARBOXYLASE GODFREY TUNNICLIFF
Laboratory
of Neurochemistry, Indiana University School of Medicine, 8600 University Boulevard, Evansville, IN 47712, U.S.A. [Tel. (812) 464-18311 (Received 6 April 1990)
INTRODUCTION Since 1950 it has been known that y-aminobutyric acid (GABA) is abundant in the brain (Awapara
et al., 1950; Roberts and Frankel, 1950; Undenfriend, 1950). It is now widely accepted that GABA carries out an important function in central nervous system activity as an inhibitory transmitter substance. By activating certain postsynaptic receptors, GABA opens Cl- channels, the outcome of which is an increase in intracellular Cl- concentration, a process which leads to a hyperpolarization and a consequent increase in the threshold for firing of the neuron (Kmjevic, 1974). This inhibitory amino acid is made in specific nerve endings by the activity of L-glutamate decarboxylase (GAD; EC 4.1.1.15), a pyridoxal phosphate-dependent enzyme occupying the rate-limiting step in GABA metabolism [see review by Tunnicliff and Ngo (198611. This enzyme has been fairly well characterized; for instance, GAD from rat brain has a molecular mass of 120 kDA and consists of two subunits of 80 and 40 kDa, respectively (Denner et al., 1987). On the other hand, the enzyme from pig brain appears to exist in three kinetically distinct forms each of which exhibits a molecular mass of 60 kDa @pink et al., 1987). Recently, the nucleotide sequence of both feline GAD cDNA (Kobayashi et al., 1987) and rat GAD cDNA (Julien et al., 1990) has been determined. Many studies have been carried out on the effects of inhibitors on GAD activity both in vivo and in vitro. The reasons for such inquiry are two-fold. First, a thorough understanding of the catalytic properties of GAD can tell us about the intracacies of the biosynthesis of GABA in the normally functioning brain. Second, the role of GABA itself can be investigated by altering its concentrations in the central nervous system. Indeed, changes in GABA function have been linked to such disorders as epilepsy, hypertension and depression (Lloyd et al., 1984; Gillis et al., 1988; Matsumoto, 1989; Motohashi er al., 1989). These studies form the basis of this review. GLUTAMATE ANALOGUES
AS COMPETITIVE INHIBITORS
In principle, compounds bearing a structural resemblance to L-glutamate can act as inhibitors of
GAD by competing with the substrate for binding at the active site. Competitive inhibitors form dead-end complexes with the enzyme that can only dissociate to the original enzyme and inhibitor: E+I kl‘EI k-1 E and I represent the enzyme and inhibitor concentrations, respectively. The concentration of EI can be described by an equilibrium constant, Ki = k-,/k,, the inhibition constant, the units of which are mol/l. Obviously the lower the K, value, the more effective the inhibitor. In practice a number of such GAD inhibitors are recognized, a few of which are extremely effective. By far the most potent of these inhibitors has been identified as 4,5-dihydroxyisophthalic acid, isolated from a Streptomyces species (Endo et al., 1978). This compound exhibited a K, of 0.18 PM with respect to the bovine enzyme. To put this inhibition in perspective, the concentration of substrate required to achieve half maximum velocity of purified enzyme is ca 0.7 mM (Wu et al., 1973). Thus if the K,,, is a reasonable measure of substrate-enzyme affinity, 4,5dihydroxyisophthalic acid has a 5000-times greater affinity for GAD than does L-glutamate. Similar conformationally restricted analogues also exhibit potent inhibition. For instance, in a study using GAD from rat brain Porter and Martin (1985) identified chelidonic acid and chelidamic acid as competitive inhibitors of the enzyme, having Ki values of 1.2 and 33 PM, respectively. Several other structurally related compounds were also shown to be good inhibitors, including 2,6-pyridine dicarboxylic acid, 3,4-dihydroxybenzoic acid and 3,4,5-trihydroxybenzoic acid. In addition, it has recently been shown in this laboratory that 3,5-pyrrazole dicarboxylic acid, another conformationally restricted substrate analogue, can compete with glutamate for the active site of mouse brain GAD with a high degree of potency (Ki = 13 PM; see Fig. 1). In earlier experiments, Tabener et al. (1977) observed that several ghitamate analogues exhibited competitive inhibition of mouse brain GAD, the most effective being thiolmalate (K, = 12 FM). Other inhibitors were at least loo-times weaker; these included malate, L-glutamate, glutamate y-hydroxamate, L-aspartate fi-hydroxamate and o-aspartate
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GODFREY TUNNICLIFF
Fig. 1. Effects of 3,5-pyrrazole carboxylic acid on partially purified mouse brain GAD. Data shown as an EadieHofstee plot. +, Absence of inhibitor; 0, presence of 25 PM inhibitor; A, presence of 50pM inhibitor. See Tunnicliff and Ngo (1978a) for assay conditions. Data replotted by method of Dixon (1953) from which a K, of 13FM was calculated (plot not shown). v = nmol/min/mg protein, s = mM glutamate.
/I-hydroxamate. The phosphonic analogue, DL-4amino-4-phosphobutyrate, is a competitive inhibitor (Lacoste et al., 1985), as are D-glutamate, DLhydroxyglutamate, DL-methylglutamate and certain Kreb’s cycle intermediates (Wu and Roberts, 1974). Tunnicliff and Ngo (1977a) noted that DL-homocysteine acted as a competitive inhibitor of GAD from mouse brain; like other carbonyl-trapping agents, however, it also inhibited GABA aminotransferase, another pyridoxal phosphate-dependent enzyme. This sulphur amino acid had previously been reported to induce convulsions after an intraperitoneal injection (Folbergrova, 1974). Another very potent competitive inhibitor is 3-mercaptopropionic acid, first shown by Lamar (1970) to inhibit GAD. i.p. Administration (32mg/kg) led to tonic-clonic seizures in rats. The closely related 4-mercaptobutyric acid also inhibited GAD and induced seizures, but not as potently. Subsequently, Wu and Roberts (1974) examined the effects of 3-mercaptopropionic acid and related mercapto acids on purified mouse brain enzyme. Ki values of 1.8, 53 and 330 p M .were obtained for 3-mercaptopropionic, 2-mercaptopropionic and 2-mercaptoacetic acids, respectively. Allylglycine is known to cause generalized seizures in experimental animals (Schneider et al., 1960) but its mechanism of action has not been fully established. Alberici et al. (1969), for example, observed inhibition of rat brain GAD both after an i.p. injection and after addition of the drug to the assay medium. Rodriguez de Lores Arnaiz et al. (1971) suggested that allylglycine competed for glutamate binding at the active site. Further, Fisher and Davies (1974) reported that this inhibitor produced curvilinear Lineweaver-Burk plots of its (comparatively weak) effects on guinea pig brain GAD. The kinetics were considered to support the concept that allylglycine acts as an irreversible inhibitor. Horton and Meldrum (1973), on the other hand, found minimal
inhibition of GAD in vitro even though the enzyme was substantially inhibited after i.p. administration. Many of these observations are consistent with the idea that a metabolite of allyglycine is actually responsible for the inhibition of GAD and the induction of convulsions. Indeed, Orlowski et al. (1977) have demonstrated that 2-keto-4pentenoic acid is a metabolite and that it is a very effective inhibitor of the enzyme. At about the same time Horton and Meldrum (1977) reported that i.v. administration of this metabolite to mice induced seizures. Later in vitro studies showed that 2-keto-4pentenoic acid competes with glutamate for binding at the active site of the enzyme (K, = 2.4 PM) (Reingold and Orlowski, 1979). GABA itself is able to inhibit GAD. The inhibition is comparatively weak but may have important physiological implications (Porter and Martin, 1984). Furthermore, there appear to be two mechanisms underlying this inhibition-one a reversible competition for substrate binding, and the second an inactivation, both the K, for the competitive inhibition and the K,,, for the irreversible effect yielded the same value (16 mM) suggesting that the inactivation step was rate-limiting (see discussion under A$inity labels). The feedback regulation of GABA synthesis would not be a unique process since other neurotransmitter production is regulated by endproduct inhibition (Ames et al., 1978). Another naturally occurring glutamate analogue, folic acid, is concentrated at nerve endings (Bridgers and McClain, 1971) and has been reported to inhibit mouse brain GAD in a competitive manner with respect to glutamate (Tunnicliff and Ngo, 1977b). These observations could reflect the involvement of this essential nutrient in the regulation of GABA synthesis. The phytopathogenic bacteria Pseudomonas tabaci produces a toxin that induces tonic-clonic convulsions in rodents (Sinden et al., 1969). The active compound appears to be a -lactylamio-/I-hydroxy-c aminopimelic acid (Wolley et al., 1952, 1955). This amino acid decreases rat brain GAD activity in vitro, the kinetics of which are consistent with competitive inhibition (Lamar and Imm, 1969). The GABA structural analogue hydrazinopropionic acid is a very strong inhibitor of mammalian GABA aminotransferase both in vitro and after i.p. injection. This compound also had an effect on GAD activity in the form of a competitive inhibition (K, = 3.4 PM). After parental administration to mice (20 mg/kg) hydrazinopropionic acid reduced GAD activity by 25% (Van Gelder, 1968).
INHIBITORS INTERFERING WITH THE COFACTOR FUNCTION
Compounds inhibiting GAD by altering pyridoxal phosphate activity are of two types: (i) those forming a complex with the cofactor (e.g. carbonyl-trapping agents); and (ii) those interfering with pyridoxal phosphate binding to the active site. Many compounds of the first type have been identified, some of which are also analogues of
GAD inhibitors L-glutamate. Accordingly they may exhibit two distinct mechanisms of action-one by competing with substrate binding to the enzyme, and the other by rendering the cofactor unavailable to participate in the d~ar~xylation catalyzed by the holoenzyme. Other inhibitors work in the intact animal by probably inhibiting pyridoxal kinase, the enzyme responsible for pyridoxal phosphate synthesis. In an important study investigating the effects of convulsant hydrazides on brain GABA metabolism, Killam and Bain (1957) observed that semicarbazide, thio~micarb~ide, isoniazid and furoyl hydrazide each inhibited rat brain GAD in vivo and in vitro, and that pyridoxal 5’-phosphate was able to reverse these effects. In addition, it has been found that semicarbazide, thiosemicarbazide, isonicotinic acid and thiocarbohydrazide all inhibited chick brain GAD activity both in vivo and in vitro (Wood and Abrahams, 19’71). In mouse brain the following hydrazides induced convulsions and inhibited GAD: thiocarbohydrazide, thiosemicarbazide, semicarbazide, carbohydrazide and isonicotinic acid hydrazide (Baltzer et al., 1960). Intramuscular injection of isonicotinamide hydrazide (INH) to chicks produces seizures and a ~oncommitant reduction in both GAD and GABA aminotransferase activity. Brain GABA concentration were initially decreased but this was followed by an elevation after a few hours (Wood and Peesker, 1972). When pyridoxine was administered with the INH, seizures were prevented as was the inhibition of GAD. However, GABA aminotransferase was still in~bited and GABA levels remained elevated. Wood and Peesker (1974) demonstrated hydrazine produced seizures in mice and that they could be antagonized by the administration of pyridoxine. Bayon et al. (1977) synthesized N-(S-phosphopyridoxyl)glutamate as a stable adduct of substrate and cofactor, and demonstrate that the compound was a competitive inhibitor of rat brain GAD with respect to pyridoxal phosphate. Choi and Churchich (1986) confirmed this observation using purified enzyme from pig brain, and reported a Ki value of 10 PM. In an earlier, extensive study of mouse brain GAD, Roberts and Simonsen (1963) reported several carbonyl-trapping agents could inhibit the enzyme. The most potent inhibitors were aminooxypropionic acid and aminooxyacetic acid, with IC, values ca 0.5 and 2 PM, respectively. For the enzyme assay the concentration of substrate present was 98 mM, while that of the cofactor was 0.08 mM. (+)ZAminooxypropionic acid and (-)2-aminooxypropionic acid were also reported to be good inhibitors. Tapia and Awapara (1969) prepared several derivatives of pyridoxal phosphate and reported that only the oxime-O-acetic acid derivative inhibited mouse brain GAD in vitro. It was later reported to show noncompetitive inhibition with respect to pyridoxal phosphate (Tapia and Sandoval, 1971). However, this compound had no appreciable effect on the enzyme after an i.p. injection at 220pmol/kg. A logical interpretation of this effect is that the compound split into its component parts in the intact animal since it has already been established that simultaneous administration of pyridoxal phosphate and amino-oxyacetic acid was without effect on GAD
1237
activity (Tapia et al., 1967). On the other hand, whereas they have no effect in oitro, the L-glutamylhydrazone and hydrazone derivatives both induced convulsions after i.p. a~inistration and inhibited GAD in viva. These drugs decrease both GAD activity and pyridoxal phosphate concentrations in the whole brain and in synaptosomal preparations (Tapia et al., 1969; Perez de la Mora et al., 1973). These authors concluded that the mechanism of GAD inhibition was via a direct inhibition of pyridoxal kinase which resulted in suboptimal cofactor concentrations for GAD activity. DL-Penicillamine when injected into mice (0.3 mg/kg) caused generalized convulsions. This effect could be prevented by the administration of pyridoxine (Matsuda and Makino, 1961). In these same penacillamine-treated animals GAD was inhibited, an effect preventable by pyridoxine. In vitro DL-~~~illa~ne inhibited GAD. Either pyridoxal 5’-phosphate or glutamate could overcome the inhibition. Abe and Matsuda (1976) administered hydrazine, rn,-penicillamine, and 2-methyl-6-amino5-hydroxymethylpyrimidine to mice. Each inhibited brain GAD although GABA concentrations were hardly altered. Another ~rbonyl-trapping agent, hydroxylamine was reported to give a competitive inhibition of mouse brain GAD with respect to substrate (Roberts and Simonsen, 1963). INACI’IVATORS
Inhibitors which irreversibly attach to an enzyme give rise to an inactivation. In reality certain of these inactivators first reversibly bind to the enzyme to form an enzyme-inhibitor complex. The reactive inhibitor can then form a covalent bond with neighbouring amino acid residues in what is an irreversible, rate-listing step: EI*E-I. I Compounds producing this type of irreversible inhibition are known as affinity labels. Ideally they are designed to structurally mimic the substrate and to possess a reactive group. Once at the active site these compounds form covalent bonds with adjacent amino acid residues. A measure of the effectiveness of these irreversible inhibitors is the inactivation constunz (&,& the concentration of inhibitor required to produce a 50% maximum rate of inactivation. This inactivation usually obeys pseudo first order kinetics. The K&, equals (k, + ~~)~~,; consequently it is larger than the Kj when k, is only partially rate-limiting. 3-Bromopyruvate is an example of an affinity label for GAD because of its resemblance to glutamate and its possession of a reactive hologen moiety. Not only does it act as a competitive inhibitor of mouse brain holoGAD (Ki = 4.2 mM), it also inactivates apoGAD in a time~e~ndent manner (Kinma= 100 mM). This inactivation does not occur in the presence of pyridoxal S-phosphate, suggesting bromopyruvate exerts its effects at the active site (Tunnicliff and Ngo, ,1978a). On a number of occasions, with various proteins, this inhibitor has been shown to alkylate cysteine residues [e.g. Birchmeier and Christen (1974)] E+I+
1238
GODPREY
?%NNlCUFF
including such residues in GAD isolated from E. cola’ Maycock, 1976). An inactivation constant (4) can be calculated for these inhibitors but this constant is not (Fonda, 1976). Therefore its action on brain GAD the same as the pi,,, for the affinity labels: indicates the presence of SH groups are necessary for enzyme activity. EI’ S-E-I E+I kl”EIk2k-t Residue-specijic inhibitors k31 E+I’ Inhibitors which react with essential amino acid residues but which cannot be considered structural The KI = Kim, [(k3 + & )l(k2 + kdlanalogues nevertheless have proven to be very useful A few mechanism-based inactivators of GAD have in the identification of amino acid residues which been described. ~Aminohex-S-ynoic acid, for exmight be involved in the catalytic process (Means and ample, has been shown to irreversibly inhibit both Feeney, 1971). These compounds, since they often do bacterial and mammalian brain GAD (Jung et al., not have an aiIinity for the active site usually produce 1978). This compound is a closer analogue of GABA an inactivation by virtue of a nonspecific bimolecular than of glutamate. Not surprisingly, therefore, the reaction exhibiting second order kinetics, i.e. the inhibitor also inactivates GABA aminotransferase existence of an EI complex is not easy to establish: (Jung and Metcalf, 1975). Another suicide inhibitor of GAD is 2-methyl-3,4-~dehydro~utamic acid E+iAE-I. which was reported to irreversibly inhibit the chick In this case a Ki,,, cannot be calculated. Phenylembryo enzyme (Chrystal et al., 1979). Both t_-glutaglyoxal is used extensively as a tool to study the mate and n-glutamate were able to reduce the degree functional role of arginine residues at the active site of inactivation, suggesting that the inhibition of various enzymes. The inhibitor binds irreversibly occurred at the active site. The measured K, was to enzymes by means of covalent bond formation 0.66mM. Rando (1979) also reported that 2-t+ with the gua~dino moiety of arginine (Takahas~, fluoromethyl glutamate is a m~hanism-baa inacti1968). Phenylgloxal irreversibly inhibits GAD from vator of GAD, the mode of action of which may be both mouse brain and E. co/i, an effect that can be dependent on the involvment of pyridoxal phosphate. antagonized by the presence of glutamate (Tunnicliff and Ngo, 1978b). A likely explanation is that an MISCELLANEOUS INHIBITORS essential arginine is at or near the active site. Both organic and inorganic mercury compounds A number of GAD inhibitors do not fit into any of can inhibit GAD activity ~unnicliff and Wood, the categories listed above. One such inhibitor is 1973, 1974). Indeed it is well established that mercury ATP, first reported to inhibit the enzyme by Tursky compounds inactivate enzymes by forming covalent (1970). A thorough examination of the mechanism of bonds with essential cysteine residues. Using a this effect has revealed that the nucleotide induces a purified GAD from mouse brain Wu and Roberts time-dependent, reversible inactivation of GAD, an (1974) discovered that p-chloromercuribenzoate action that could very well have significance as far as (PCMB) was a powerful inhibitor when present regulation of GABA synthesis is concerned (Martin, during the assay and that this inhibition was dimin1987)‘ ished as substrate concentration was increased. No Several divalent metal ions have been shown to kinetic constant was reported. 5,5’-Dithiobis(Zniinhibit mouse brain GAD, the most potent being trohenzoic acid) (DTNB) was also reported to be a Zn2+ (Wu and Roberts, 1974). It has since been potent inhibitor of the enzyme and produced an demonstrated that this inhibition was competitive apparent competitive inhibition with a K, value of with respect to glutamate, with a Ki = 55 pM (Tunni11 nM, easily the lowest reported of any inhibitor of cliff and Ngo, 1978c). Three distinct Zn’+-binding this enzyme. Although both PCMB and DTNB are proteins have been isolated from rat brain and it has known to be irreversible inhibitors of many enzymic been proposed that they may alter Zn*+ levels and reactions by reacting with essential cysteine residues thus indirectly modulate GAD activity (Itoh et al., (Means and Feaney, 1971), Wu and Roberts did not 1983; Ebadi et al., 1984). investigate the possibility of an inactivation process Exposure of GAD to oxygen at high pressure in their enzyme. Since both these compounds are before measurement of enzyme activity has been known to react with SH groups, this is further shown to adversely affect activity (Tunnicliff et al., evidence for the presence of essential cysteine residues 1973; Tun~cliff and Wood, 1973). Furthermore, at the active site of GAD. exposure of the enzyme to high pressure oxygen during the measurement of activity also inhibits Mechanism -based inhibitors the enzyme (Tunnicliff and Wood, 1974). It seems reasonable to assume that oxygen, mercury comSome inhibitors rely on the enzyme to convert pounds, DTNB and 3-bromopyruvate each inhibit them to a reactive species. These are termed suicide or m~ha~srn-based inactivators (Silverman, 1988). the enzyme by reacting with essential cysteine residues. Such compounds are in actuality substrates for the target enzyme, the catalytic activity of which produces an inhibitory metabolite at the active site. SIGNIFICANCE Generally these reactive products form covalent The evidence that GABA is associated with several bonds with essential residues. Sometimes, however, disorders involving the central nervous system is they bind to the enzyme with such high affinity a~~uiating and many neuroscientists believe that that they appear to act as inactivators (Abeles and
GAD inhibitors
this important inhibitory amino acid has a role to play in epilepsy, hypertension, anxiety and perhaps depression (Matsumoto, 1989). Since GAD is the key enzyme in the biosynthesis of GABA at nerve endings, any interference in its catalytic activity could be expected to have profound results in the intact animal. A stark illustration of this is the induction of generalized seizures by GAD inhibitor. More subtle effects at lower doses of inhibitors have also been observed. For instance, 3-mercaptopropionic acid can influence blood pressure in guinea pigs (Alsip er at., 1984). As documented in this review, there are many compounds capable of inhibiting isolated GAD and their actions can often reveal valuable information about the enzyme. For instance, the inactivation by SH-group reagents suggests the existence of cysteine residues necessary for catalytic activity, and the inhibition by phenylglyoxal indicates there are essential arginine residues. For those researchers interested in studying the effect of specific inhibitors in the intact animal, however, compounds capable of com~ting with L-glutamate as structural analogues offer the best strategy. It appears that the most effective analogues are those of restricted conformation exemplified by 3,4-dihyroxyisoph~alic acid. The fact that the most effective substrate analogue inhibitors are of restricted confirmation is good evidence that L-glutamate as substrate binds to the active site of GAD in its extended form. Unfortunately such inhibitors often cannot penetrate the blood-brain barrier. Sometimes the direct introduction of the inhibitor into the brain can overcome this disadvantage although this technique has several drawbacks, including uneven distribution in the brain.
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Rando R. R. (1979) Specific inhibitors of GABA metabolism. In GABA-Neurotransmifters (Edited by Krogsgaard-Larsen P., Scheel-Kruger J. and Hofod H.), pp. 228-235. Munksgaard, Copenhagen. Reingold D. F. and Orlowski M. (19X9) Inhibition of brain glutamate decarboxylase by 2-keto4pentenoic acid, a metabolite of allylglycine. J. Neurochem. 32, 907-913.
Roberts E. and Frankel S. (1950) y-Aminobutyric acid in brain: its formation from glutamic acid. J. biol. Chem. 187, 55-63. Roberts E. and Simonsen D. G. (1963) Some properties of tglutamic decarboxylase in mouse brain. Biochem. Pharmac. 12, 113-134. Rodriguez de Lores Amaiz G., Alberici de Canal M. and De Robertis E. (1971) 2-Amine 4pentenoic acid (allylglycine): a proposed tool for the study of GABA mediated systems. Int. J. Neurosci. 2, 137-144. Schneider J. H., Cassir R. and Chordikian F. (1960) Inhibition of incorporation of thymidine into deoxyribonucleic acid by amino acid antagonists in viuo. J. biol. Chem. 235, 1437-1440. Silverman R. B. (1988) Mechanism-Based Enzyme Inactivation: Chemistry and Enzymology, Vols 1 and 2. CRC Press, Boca Raton, Fla.
Sinden S. L., Durbin R. D., Uchytil T. F. and Lamar C. (1969) The production by an exotoxin from Pseudomonas tabaci. Toxic. Appi. Pharmac.
14, 82-88.
Spink D. C., Porter T. G., Wu S. J. and Martin D. L. (1987) Characterization of three kinetically distinct forms of glutamate decarboxylase from pig brain. Biochem. J. 231, 695-703. Tabemer P. V., Pearce M. J. and Watkins J. C. (1977) The inhibition of mouse brain glutamate decarboxylase by some structural analogues of L-glutamate acid. Biochem. Pharmac. 26, 345-349.
Takahashi K. (1968) The reaction of phenylglyoxal with arginine residues in proteins. J. biol. Chem. 243, 6171-6179. Tapia R. and Awapara J. (1969) Effects of various substituted hydrazones and hydraxines of pyridoxal-5’-phosphate on brain glutamate decarboxylase. Biochem. Pharmac. 1% 145-152. Tapia R. and Sandoval M. E. (1971) Study on the inhibition of brain glutamate dccarboxylase by pyridoxal phosphate oxime-O-acetic acid. J. Neurochem. 18, 2051-2059.
Tapia R., Perez de la Mora M. and Massieu G. H. (1967) Modifications of brain glutamate decarboxylase activity by pyridoxal phosphate-gamma-glutamyl hydrazone. Biochem. Pharmac.
16, 1211-1218.
Tunnicliff G. and Ngo T. T. (1977a) The mode of action of homocysteine on mouse brain glutamic decarboxylase and y-aminobutyrate aminotransferase. Can. J. Biochem. 55, 1013-1018.
Tunnicliff G. and Ngo T. T. (1977b) Folic acid and the inhibition of brain glutamic acid decarboxyalse. Experientia 33, 67-68.
Tunnicliff G. and Ngo T. T. (1978a) Mechanism of inactivation of brain glutamic decarboxylase by 3-bromopyruvate. Int. J. Biochem. 9, 249-252. Tunnicliff G. and Ngo T. T. (1978b) Functional role of arginine residues in glutamic acid decarboxylase from brain and bacteria. Experientia 34, 989-990. Tunnicliff G. and Ngo T. T. (1978~) Competitive inhibition of mouse brain glutamic decarboxylase by zinc ions. IRCS Med. Sci. 6, 45 1. Tunnicliff G. and Ngo T. T. (1986) Regulation of y-aminobutyric acid synthesis in the vertebrate nervous system. Neurochem. I&. 8, 287-297. Tunnicliff G. and Wood J. D. (1973) The inhibition of mouse brain neurotransmitter enzymes by mercury compounds and a comparison with the effects of hyperbaric oxygen. Comp. gen. Pharmac. 4, 101-105. Tunnicliff G. and Wood J. D. (1974) Influence of hyperbaric oxygen upon transmitter enzymes of chick brain. ht. J. Biochem. 5, 555-559.
Tunnicliff G., Urton M. and Wood J. D. (1973) Susceptibility of chick brain L-glutamate decarboxylase and other neurotransmitter enzymes to hyperbaric oxygen in vitro. Biochem. Pharmac.
22, 501-505.
Tursky T. (1970) Inhibition of brain glutamate decarboxylase by adenosine triphosphate. Eur. J. Biochem. 12, 544-549.
Udenfriend S. (1950) Identification of y-aminobutyric acid in brain by the isotope derivative method. J. biol. Chem. 187, 65-69. Van Gelder N. M. (1968) Hydraxinopropionic acid: a new inhibitor of aminobutvrate transaminase and glutamate decarboxylase. J. Neurochem. 15, 747-757. Wood J. D. and Abrahams D. E. (1971) The comparative effect of various hydraxides on y-aminobutyric acid and its metabolism. J. Neurochem. 18, 1017-1025. Wood J. D. and Peesker S. J. (1972) The effect on GABA metabolism in brain of isonicotinic acid hydrazide and pyridoxine as a function of time after administration. J. Neurochem. 19, 1527-1537.
GAD inhibitors Wood J. D. and Peesker S. J. (1974) Development of an expression which relates the excitable state of the brain to the level of GAD activity and GABA content, with particular reference to the action of hydrazine and its derivatives. J. Neurochem. 23, 703-712. Wooley D. W., Pringle R. B. and Braun A. C. (1952) Isolation of the phytopathogenic toxin of Pseudomonas rabaci, an antagonist of methionine. J. biol. Chem. 197. 409.
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Wooley D. W., Schaffner G. and Braun A. C. (1955) Studies on the structure of the phytopathogenic toxin of Pseudomonas tabaci. J. biol. Chem. 215, 485.
Wu J-Y. and Roberts E. (1974) Properties of brain L-glutamate decarboxylase: inhibition studies. J. Neurochem. 23, 759-767. Wu J-Y., Matsuda T. and Roberts E. (1973) Purification and characterization of glutamate decarboxylase from mouse brain. J. biol. Chem. 248. 3029-3034.
fnr. J. Biochem.Vol. 22, No. 11, pp. 1235-1241,1990 Printed in Great Britain. All rights reserved
MINIREVIEW ACTION
OF INHIBITORS ON BRAIN GLUTAMATE DECARBOXYLASE GODFREY TUNNICLIFF
Laboratory
of Neurochemistry, Indiana University School of Medicine, 8600 University Boulevard, Evansville, IN 47712, U.S.A. [Tel. (812) 464-18311 (Received 6 April 1990)
INTRODUCTION Since 1950 it has been known that y-aminobutyric acid (GABA) is abundant in the brain (Awapara
et al., 1950; Roberts and Frankel, 1950; Undenfriend, 1950). It is now widely accepted that GABA carries out an important function in central nervous system activity as an inhibitory transmitter substance. By activating certain postsynaptic receptors, GABA opens Cl- channels, the outcome of which is an increase in intracellular Cl- concentration, a process which leads to a hyperpolarization and a consequent increase in the threshold for firing of the neuron (Kmjevic, 1974). This inhibitory amino acid is made in specific nerve endings by the activity of L-glutamate decarboxylase (GAD; EC 4.1.1.15), a pyridoxal phosphate-dependent enzyme occupying the rate-limiting step in GABA metabolism [see review by Tunnicliff and Ngo (198611. This enzyme has been fairly well characterized; for instance, GAD from rat brain has a molecular mass of 120 kDA and consists of two subunits of 80 and 40 kDa, respectively (Denner et al., 1987). On the other hand, the enzyme from pig brain appears to exist in three kinetically distinct forms each of which exhibits a molecular mass of 60 kDa @pink et al., 1987). Recently, the nucleotide sequence of both feline GAD cDNA (Kobayashi et al., 1987) and rat GAD cDNA (Julien et al., 1990) has been determined. Many studies have been carried out on the effects of inhibitors on GAD activity both in vivo and in vitro. The reasons for such inquiry are two-fold. First, a thorough understanding of the catalytic properties of GAD can tell us about the intracacies of the biosynthesis of GABA in the normally functioning brain. Second, the role of GABA itself can be investigated by altering its concentrations in the central nervous system. Indeed, changes in GABA function have been linked to such disorders as epilepsy, hypertension and depression (Lloyd et al., 1984; Gillis et al., 1988; Matsumoto, 1989; Motohashi er al., 1989). These studies form the basis of this review. GLUTAMATE ANALOGUES
AS COMPETITIVE INHIBITORS
In principle, compounds bearing a structural resemblance to L-glutamate can act as inhibitors of
GAD by competing with the substrate for binding at the active site. Competitive inhibitors form dead-end complexes with the enzyme that can only dissociate to the original enzyme and inhibitor: E+I kl‘EI k-1 E and I represent the enzyme and inhibitor concentrations, respectively. The concentration of EI can be described by an equilibrium constant, Ki = k-,/k,, the inhibition constant, the units of which are mol/l. Obviously the lower the K, value, the more effective the inhibitor. In practice a number of such GAD inhibitors are recognized, a few of which are extremely effective. By far the most potent of these inhibitors has been identified as 4,5-dihydroxyisophthalic acid, isolated from a Streptomyces species (Endo et al., 1978). This compound exhibited a K, of 0.18 PM with respect to the bovine enzyme. To put this inhibition in perspective, the concentration of substrate required to achieve half maximum velocity of purified enzyme is ca 0.7 mM (Wu et al., 1973). Thus if the K,,, is a reasonable measure of substrate-enzyme affinity, 4,5dihydroxyisophthalic acid has a 5000-times greater affinity for GAD than does L-glutamate. Similar conformationally restricted analogues also exhibit potent inhibition. For instance, in a study using GAD from rat brain Porter and Martin (1985) identified chelidonic acid and chelidamic acid as competitive inhibitors of the enzyme, having Ki values of 1.2 and 33 PM, respectively. Several other structurally related compounds were also shown to be good inhibitors, including 2,6-pyridine dicarboxylic acid, 3,4-dihydroxybenzoic acid and 3,4,5-trihydroxybenzoic acid. In addition, it has recently been shown in this laboratory that 3,5-pyrrazole dicarboxylic acid, another conformationally restricted substrate analogue, can compete with glutamate for the active site of mouse brain GAD with a high degree of potency (Ki = 13 PM; see Fig. 1). In earlier experiments, Tabener et al. (1977) observed that several ghitamate analogues exhibited competitive inhibition of mouse brain GAD, the most effective being thiolmalate (K, = 12 FM). Other inhibitors were at least loo-times weaker; these included malate, L-glutamate, glutamate y-hydroxamate, L-aspartate fi-hydroxamate and o-aspartate
1235
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GODFREY TUNNICLIFF
Fig. 1. Effects of 3,5-pyrrazole carboxylic acid on partially purified mouse brain GAD. Data shown as an EadieHofstee plot. +, Absence of inhibitor; 0, presence of 25 PM inhibitor; A, presence of 50pM inhibitor. See Tunnicliff and Ngo (1978a) for assay conditions. Data replotted by method of Dixon (1953) from which a K, of 13FM was calculated (plot not shown). v = nmol/min/mg protein, s = mM glutamate.
/I-hydroxamate. The phosphonic analogue, DL-4amino-4-phosphobutyrate, is a competitive inhibitor (Lacoste et al., 1985), as are D-glutamate, DLhydroxyglutamate, DL-methylglutamate and certain Kreb’s cycle intermediates (Wu and Roberts, 1974). Tunnicliff and Ngo (1977a) noted that DL-homocysteine acted as a competitive inhibitor of GAD from mouse brain; like other carbonyl-trapping agents, however, it also inhibited GABA aminotransferase, another pyridoxal phosphate-dependent enzyme. This sulphur amino acid had previously been reported to induce convulsions after an intraperitoneal injection (Folbergrova, 1974). Another very potent competitive inhibitor is 3-mercaptopropionic acid, first shown by Lamar (1970) to inhibit GAD. i.p. Administration (32mg/kg) led to tonic-clonic seizures in rats. The closely related 4-mercaptobutyric acid also inhibited GAD and induced seizures, but not as potently. Subsequently, Wu and Roberts (1974) examined the effects of 3-mercaptopropionic acid and related mercapto acids on purified mouse brain enzyme. Ki values of 1.8, 53 and 330 p M .were obtained for 3-mercaptopropionic, 2-mercaptopropionic and 2-mercaptoacetic acids, respectively. Allylglycine is known to cause generalized seizures in experimental animals (Schneider et al., 1960) but its mechanism of action has not been fully established. Alberici et al. (1969), for example, observed inhibition of rat brain GAD both after an i.p. injection and after addition of the drug to the assay medium. Rodriguez de Lores Arnaiz et al. (1971) suggested that allylglycine competed for glutamate binding at the active site. Further, Fisher and Davies (1974) reported that this inhibitor produced curvilinear Lineweaver-Burk plots of its (comparatively weak) effects on guinea pig brain GAD. The kinetics were considered to support the concept that allylglycine acts as an irreversible inhibitor. Horton and Meldrum (1973), on the other hand, found minimal
inhibition of GAD in vitro even though the enzyme was substantially inhibited after i.p. administration. Many of these observations are consistent with the idea that a metabolite of allyglycine is actually responsible for the inhibition of GAD and the induction of convulsions. Indeed, Orlowski et al. (1977) have demonstrated that 2-keto-4pentenoic acid is a metabolite and that it is a very effective inhibitor of the enzyme. At about the same time Horton and Meldrum (1977) reported that i.v. administration of this metabolite to mice induced seizures. Later in vitro studies showed that 2-keto-4pentenoic acid competes with glutamate for binding at the active site of the enzyme (K, = 2.4 PM) (Reingold and Orlowski, 1979). GABA itself is able to inhibit GAD. The inhibition is comparatively weak but may have important physiological implications (Porter and Martin, 1984). Furthermore, there appear to be two mechanisms underlying this inhibition-one a reversible competition for substrate binding, and the second an inactivation, both the K, for the competitive inhibition and the K,,, for the irreversible effect yielded the same value (16 mM) suggesting that the inactivation step was rate-limiting (see discussion under A$inity labels). The feedback regulation of GABA synthesis would not be a unique process since other neurotransmitter production is regulated by endproduct inhibition (Ames et al., 1978). Another naturally occurring glutamate analogue, folic acid, is concentrated at nerve endings (Bridgers and McClain, 1971) and has been reported to inhibit mouse brain GAD in a competitive manner with respect to glutamate (Tunnicliff and Ngo, 1977b). These observations could reflect the involvement of this essential nutrient in the regulation of GABA synthesis. The phytopathogenic bacteria Pseudomonas tabaci produces a toxin that induces tonic-clonic convulsions in rodents (Sinden et al., 1969). The active compound appears to be a -lactylamio-/I-hydroxy-c aminopimelic acid (Wolley et al., 1952, 1955). This amino acid decreases rat brain GAD activity in vitro, the kinetics of which are consistent with competitive inhibition (Lamar and Imm, 1969). The GABA structural analogue hydrazinopropionic acid is a very strong inhibitor of mammalian GABA aminotransferase both in vitro and after i.p. injection. This compound also had an effect on GAD activity in the form of a competitive inhibition (K, = 3.4 PM). After parental administration to mice (20 mg/kg) hydrazinopropionic acid reduced GAD activity by 25% (Van Gelder, 1968).
INHIBITORS INTERFERING WITH THE COFACTOR FUNCTION
Compounds inhibiting GAD by altering pyridoxal phosphate activity are of two types: (i) those forming a complex with the cofactor (e.g. carbonyl-trapping agents); and (ii) those interfering with pyridoxal phosphate binding to the active site. Many compounds of the first type have been identified, some of which are also analogues of
GAD inhibitors L-glutamate. Accordingly they may exhibit two distinct mechanisms of action-one by competing with substrate binding to the enzyme, and the other by rendering the cofactor unavailable to participate in the d~ar~xylation catalyzed by the holoenzyme. Other inhibitors work in the intact animal by probably inhibiting pyridoxal kinase, the enzyme responsible for pyridoxal phosphate synthesis. In an important study investigating the effects of convulsant hydrazides on brain GABA metabolism, Killam and Bain (1957) observed that semicarbazide, thio~micarb~ide, isoniazid and furoyl hydrazide each inhibited rat brain GAD in vivo and in vitro, and that pyridoxal 5’-phosphate was able to reverse these effects. In addition, it has been found that semicarbazide, thiosemicarbazide, isonicotinic acid and thiocarbohydrazide all inhibited chick brain GAD activity both in vivo and in vitro (Wood and Abrahams, 19’71). In mouse brain the following hydrazides induced convulsions and inhibited GAD: thiocarbohydrazide, thiosemicarbazide, semicarbazide, carbohydrazide and isonicotinic acid hydrazide (Baltzer et al., 1960). Intramuscular injection of isonicotinamide hydrazide (INH) to chicks produces seizures and a ~oncommitant reduction in both GAD and GABA aminotransferase activity. Brain GABA concentration were initially decreased but this was followed by an elevation after a few hours (Wood and Peesker, 1972). When pyridoxine was administered with the INH, seizures were prevented as was the inhibition of GAD. However, GABA aminotransferase was still in~bited and GABA levels remained elevated. Wood and Peesker (1974) demonstrated hydrazine produced seizures in mice and that they could be antagonized by the administration of pyridoxine. Bayon et al. (1977) synthesized N-(S-phosphopyridoxyl)glutamate as a stable adduct of substrate and cofactor, and demonstrate that the compound was a competitive inhibitor of rat brain GAD with respect to pyridoxal phosphate. Choi and Churchich (1986) confirmed this observation using purified enzyme from pig brain, and reported a Ki value of 10 PM. In an earlier, extensive study of mouse brain GAD, Roberts and Simonsen (1963) reported several carbonyl-trapping agents could inhibit the enzyme. The most potent inhibitors were aminooxypropionic acid and aminooxyacetic acid, with IC, values ca 0.5 and 2 PM, respectively. For the enzyme assay the concentration of substrate present was 98 mM, while that of the cofactor was 0.08 mM. (+)ZAminooxypropionic acid and (-)2-aminooxypropionic acid were also reported to be good inhibitors. Tapia and Awapara (1969) prepared several derivatives of pyridoxal phosphate and reported that only the oxime-O-acetic acid derivative inhibited mouse brain GAD in vitro. It was later reported to show noncompetitive inhibition with respect to pyridoxal phosphate (Tapia and Sandoval, 1971). However, this compound had no appreciable effect on the enzyme after an i.p. injection at 220pmol/kg. A logical interpretation of this effect is that the compound split into its component parts in the intact animal since it has already been established that simultaneous administration of pyridoxal phosphate and amino-oxyacetic acid was without effect on GAD
1237
activity (Tapia et al., 1967). On the other hand, whereas they have no effect in oitro, the L-glutamylhydrazone and hydrazone derivatives both induced convulsions after i.p. a~inistration and inhibited GAD in viva. These drugs decrease both GAD activity and pyridoxal phosphate concentrations in the whole brain and in synaptosomal preparations (Tapia et al., 1969; Perez de la Mora et al., 1973). These authors concluded that the mechanism of GAD inhibition was via a direct inhibition of pyridoxal kinase which resulted in suboptimal cofactor concentrations for GAD activity. DL-Penicillamine when injected into mice (0.3 mg/kg) caused generalized convulsions. This effect could be prevented by the administration of pyridoxine (Matsuda and Makino, 1961). In these same penacillamine-treated animals GAD was inhibited, an effect preventable by pyridoxine. In vitro DL-~~~illa~ne inhibited GAD. Either pyridoxal 5’-phosphate or glutamate could overcome the inhibition. Abe and Matsuda (1976) administered hydrazine, rn,-penicillamine, and 2-methyl-6-amino5-hydroxymethylpyrimidine to mice. Each inhibited brain GAD although GABA concentrations were hardly altered. Another ~rbonyl-trapping agent, hydroxylamine was reported to give a competitive inhibition of mouse brain GAD with respect to substrate (Roberts and Simonsen, 1963). INACI’IVATORS
Inhibitors which irreversibly attach to an enzyme give rise to an inactivation. In reality certain of these inactivators first reversibly bind to the enzyme to form an enzyme-inhibitor complex. The reactive inhibitor can then form a covalent bond with neighbouring amino acid residues in what is an irreversible, rate-listing step: EI*E-I. I Compounds producing this type of irreversible inhibition are known as affinity labels. Ideally they are designed to structurally mimic the substrate and to possess a reactive group. Once at the active site these compounds form covalent bonds with adjacent amino acid residues. A measure of the effectiveness of these irreversible inhibitors is the inactivation constunz (&,& the concentration of inhibitor required to produce a 50% maximum rate of inactivation. This inactivation usually obeys pseudo first order kinetics. The K&, equals (k, + ~~)~~,; consequently it is larger than the Kj when k, is only partially rate-limiting. 3-Bromopyruvate is an example of an affinity label for GAD because of its resemblance to glutamate and its possession of a reactive hologen moiety. Not only does it act as a competitive inhibitor of mouse brain holoGAD (Ki = 4.2 mM), it also inactivates apoGAD in a time~e~ndent manner (Kinma= 100 mM). This inactivation does not occur in the presence of pyridoxal S-phosphate, suggesting bromopyruvate exerts its effects at the active site (Tunnicliff and Ngo, ,1978a). On a number of occasions, with various proteins, this inhibitor has been shown to alkylate cysteine residues [e.g. Birchmeier and Christen (1974)] E+I+
1238
GODPREY
?%NNlCUFF
including such residues in GAD isolated from E. cola’ Maycock, 1976). An inactivation constant (4) can be calculated for these inhibitors but this constant is not (Fonda, 1976). Therefore its action on brain GAD the same as the pi,,, for the affinity labels: indicates the presence of SH groups are necessary for enzyme activity. EI’ S-E-I E+I kl”EIk2k-t Residue-specijic inhibitors k31 E+I’ Inhibitors which react with essential amino acid residues but which cannot be considered structural The KI = Kim, [(k3 + & )l(k2 + kdlanalogues nevertheless have proven to be very useful A few mechanism-based inactivators of GAD have in the identification of amino acid residues which been described. ~Aminohex-S-ynoic acid, for exmight be involved in the catalytic process (Means and ample, has been shown to irreversibly inhibit both Feeney, 1971). These compounds, since they often do bacterial and mammalian brain GAD (Jung et al., not have an aiIinity for the active site usually produce 1978). This compound is a closer analogue of GABA an inactivation by virtue of a nonspecific bimolecular than of glutamate. Not surprisingly, therefore, the reaction exhibiting second order kinetics, i.e. the inhibitor also inactivates GABA aminotransferase existence of an EI complex is not easy to establish: (Jung and Metcalf, 1975). Another suicide inhibitor of GAD is 2-methyl-3,4-~dehydro~utamic acid E+iAE-I. which was reported to irreversibly inhibit the chick In this case a Ki,,, cannot be calculated. Phenylembryo enzyme (Chrystal et al., 1979). Both t_-glutaglyoxal is used extensively as a tool to study the mate and n-glutamate were able to reduce the degree functional role of arginine residues at the active site of inactivation, suggesting that the inhibition of various enzymes. The inhibitor binds irreversibly occurred at the active site. The measured K, was to enzymes by means of covalent bond formation 0.66mM. Rando (1979) also reported that 2-t+ with the gua~dino moiety of arginine (Takahas~, fluoromethyl glutamate is a m~hanism-baa inacti1968). Phenylgloxal irreversibly inhibits GAD from vator of GAD, the mode of action of which may be both mouse brain and E. co/i, an effect that can be dependent on the involvment of pyridoxal phosphate. antagonized by the presence of glutamate (Tunnicliff and Ngo, 1978b). A likely explanation is that an MISCELLANEOUS INHIBITORS essential arginine is at or near the active site. Both organic and inorganic mercury compounds A number of GAD inhibitors do not fit into any of can inhibit GAD activity ~unnicliff and Wood, the categories listed above. One such inhibitor is 1973, 1974). Indeed it is well established that mercury ATP, first reported to inhibit the enzyme by Tursky compounds inactivate enzymes by forming covalent (1970). A thorough examination of the mechanism of bonds with essential cysteine residues. Using a this effect has revealed that the nucleotide induces a purified GAD from mouse brain Wu and Roberts time-dependent, reversible inactivation of GAD, an (1974) discovered that p-chloromercuribenzoate action that could very well have significance as far as (PCMB) was a powerful inhibitor when present regulation of GABA synthesis is concerned (Martin, during the assay and that this inhibition was dimin1987)‘ ished as substrate concentration was increased. No Several divalent metal ions have been shown to kinetic constant was reported. 5,5’-Dithiobis(Zniinhibit mouse brain GAD, the most potent being trohenzoic acid) (DTNB) was also reported to be a Zn2+ (Wu and Roberts, 1974). It has since been potent inhibitor of the enzyme and produced an demonstrated that this inhibition was competitive apparent competitive inhibition with a K, value of with respect to glutamate, with a Ki = 55 pM (Tunni11 nM, easily the lowest reported of any inhibitor of cliff and Ngo, 1978c). Three distinct Zn’+-binding this enzyme. Although both PCMB and DTNB are proteins have been isolated from rat brain and it has known to be irreversible inhibitors of many enzymic been proposed that they may alter Zn*+ levels and reactions by reacting with essential cysteine residues thus indirectly modulate GAD activity (Itoh et al., (Means and Feaney, 1971), Wu and Roberts did not 1983; Ebadi et al., 1984). investigate the possibility of an inactivation process Exposure of GAD to oxygen at high pressure in their enzyme. Since both these compounds are before measurement of enzyme activity has been known to react with SH groups, this is further shown to adversely affect activity (Tunnicliff et al., evidence for the presence of essential cysteine residues 1973; Tun~cliff and Wood, 1973). Furthermore, at the active site of GAD. exposure of the enzyme to high pressure oxygen during the measurement of activity also inhibits Mechanism -based inhibitors the enzyme (Tunnicliff and Wood, 1974). It seems reasonable to assume that oxygen, mercury comSome inhibitors rely on the enzyme to convert pounds, DTNB and 3-bromopyruvate each inhibit them to a reactive species. These are termed suicide or m~ha~srn-based inactivators (Silverman, 1988). the enzyme by reacting with essential cysteine residues. Such compounds are in actuality substrates for the target enzyme, the catalytic activity of which produces an inhibitory metabolite at the active site. SIGNIFICANCE Generally these reactive products form covalent The evidence that GABA is associated with several bonds with essential residues. Sometimes, however, disorders involving the central nervous system is they bind to the enzyme with such high affinity a~~uiating and many neuroscientists believe that that they appear to act as inactivators (Abeles and
GAD inhibitors
this important inhibitory amino acid has a role to play in epilepsy, hypertension, anxiety and perhaps depression (Matsumoto, 1989). Since GAD is the key enzyme in the biosynthesis of GABA at nerve endings, any interference in its catalytic activity could be expected to have profound results in the intact animal. A stark illustration of this is the induction of generalized seizures by GAD inhibitor. More subtle effects at lower doses of inhibitors have also been observed. For instance, 3-mercaptopropionic acid can influence blood pressure in guinea pigs (Alsip er at., 1984). As documented in this review, there are many compounds capable of inhibiting isolated GAD and their actions can often reveal valuable information about the enzyme. For instance, the inactivation by SH-group reagents suggests the existence of cysteine residues necessary for catalytic activity, and the inhibition by phenylglyoxal indicates there are essential arginine residues. For those researchers interested in studying the effect of specific inhibitors in the intact animal, however, compounds capable of com~ting with L-glutamate as structural analogues offer the best strategy. It appears that the most effective analogues are those of restricted conformation exemplified by 3,4-dihyroxyisoph~alic acid. The fact that the most effective substrate analogue inhibitors are of restricted confirmation is good evidence that L-glutamate as substrate binds to the active site of GAD in its extended form. Unfortunately such inhibitors often cannot penetrate the blood-brain barrier. Sometimes the direct introduction of the inhibitor into the brain can overcome this disadvantage although this technique has several drawbacks, including uneven distribution in the brain.
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Alsip N. L., Simon J. R., Fohi L. D. and DiMicco J. A. (1984) Cardiova~~ar effects of 3-mer~ptopropionic acid and levels of GABA in regions of tbe brain of guinea-pigs. Neuropharmacology 23, 349-351. Ames M. M., Lemer P. and Lover&erg W. (1978) Tyrosine hydroxylase: activation by protein phosphorylation and endproduct inhibition. J. biof. Chem. 253, 27-31. Awapara J., Landau A. J., Furest R. and Seale B. (1950) Free y-aminobutyric acid in brain. J. biol. Chem. 187, 35-39. Balzer H., Holtz P. and Palm D. (1960) Untersuchungen iiber die bi~hemi~hen Grundlagen der konvulsiv~ Wirkung von Hydraziden. Naunyn-Schmiedberg’s Arch. exp. Path. Pharmak. 239, 520-552.
Bayon A., Possani L. D. and Tapia R. (1977) Kinetics of brain glutamate decarboxylase. Inhibition studies with N-(5’-phosphopy~doxal) amino acids. f. Neurochem. 29, 513-517. Birchmeier W. and Christen P. (1974) The reaction of cytoplasmic aspartate aminotransferase with bromo-
1239
uyruvate. Syncatalytic modification stimulates affinity h&eling. J. biol. Chem. 249, 6311-6315. Bridges W. F. and M&lain L. D. 11971) Some interrelationshrps of pyridoxal phosphate, fohc acid and serine metabolism in brain. Ado. Biochem. Psychopharmac. 4, 81-92.
Choi S. Y. and Churchich J. E. (1986) Glutamate decarboxylase side reactions catalyzed by the enzyme. Eur. J. Biochem. 160, 5 1S-520.
Chrystal E,, Bey P. and Rando R. R. (1979) The irreversible inhibition of brain L-glutamate-l-decarboxylase by (2RS,3E)-2-methyl-3,4-didehydroglutamic acid. J. Neuro&em. 32, 1501-1507. Denner L. A., Wei S. C., Lin H. S., Lin C-T. and Wu J-Y. (1987) Brain L-glutamate decarboxylase: purification and subunit structure. Proc. natn. Acad. Sci. U.S.A. 84, 668-672.
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