Biochemical consequences of reactions catalyzed by GAD and GABA-T

Brain Reserrrch

Rul~eti/i,

Vol. 5,

Suppl. 2, pp. 375-379.

Printed in the U.8.A

Biochemical Consequences of Reactions Catalyzed by GAD and GABA-T BRUCE

LIPPERT”‘, Resenrch

~~~rr~~l Wentre

de Recherche

MICHEL

J. JUNG?

AND

BRIAN

W. METCALF*

Center,

2110 E. ~~lbr~l~th Rd., ~incin~~t~, QH 45215 and Merrell International, 16 rue d’Ankaru, 67084 Strasbourg, Cedex,

France

LIPPERT, B., M. J. JUNG AND B. W. METCALF. ~~~ch~rni~ltl ~~)n.~~qu~~~e~ctf rmctions caruly,-rd by GAD und GABA-T. BRAIN RES. BULL. 5: Sued. 2. 375-379. 1980.-GABA-T abstracts the nro-4-S proton from GABA. The enzyme abstracts the analogous protons from (+)-y-acetyienic GABA and (+)-y-vinyl GABA in each case precipitating its own inactivation. (+)-y-Acetylenic GABA also irreversibly inhibits GAD indicating a transamination capability of the decarboxylase on this synthetic GABA analogue. y-Vinyl GABA has no effect on this enzyme in vifro. Administration of either y-acetylenic or y-vinyl GABA to rats or mice results in a dose-dependent increase in brain GABA levels. At any given level of GABA-T inhibition the concentrations of GABA are higher after y-vinyl GABA due to its smaller effect on GAD. Nevertheless, after a dose of y-vinyl GABA sufficient to raise brain GABA levels to over 300% of control levels for 48 hours, there is a slow decrease in brain GAD activity to 75% and 65% of control levels at 24 and 48 hours respectively. This diminution of GAD activity after administration of y-vinyl GABA is consistent with a feedback effect of sustained elevated GABA levels on the synthesis of GAD. II

GABA

y-Acetylenic

GABA

,

y-Vinyl GABA

GABA-T

4-AMI~GBUTYRI~ ACID (GABA) is believed to be an important inhibitory neurotransmitter in mammalian brain [ 131. It is formed from L-glutamate in a reaction catalyzed by l-L-glutamate carboxylyase (GAD) and is metabolized mainly by transamination with a-ketoglutarate in a reaction catalyzed by 4-~inobuty~c acid: a-oxoglutarate aminotransferase (GABA-T). A decrease in GAD activity or GABA function in discrete areas of the brain has been demonstrated or implicated in such diverse diseases as epilepsy [ 181, schizophrenia 1271, Huntington’s disease [24], Parkinsonism [17] and tardive dyskinesias 123,241. Therefore, pharmacological manipulation of the GABA system to increase the availability of this neurotransmitter at its receptors in the affected tracts could have useful therapeutic effects. No means has yet been devised to increase GABA synthesis. nor have specific, non-toxic inhibitors of GABA re-uptake which readily cross the blood-brain barrier been found. Furthermore, peripherally administered GABA does not reach the brain in mature animals. Our efforts, therefore, have been devoted to the specific inhibition of GABA catabolism through the design, synthesis and use of enzyme-activated irreversible inhibitors of GABA-T [9, 16, 21, 221. Previous inhibitors of GABA-T, such as aminoxyacetic acid [3] or n-dipropyla~e~te [29], were respectively much less selective and thereby toxic or of low potency and short duration of action. Ethanolamine-O-sulfate [5,61, the first selective irreversible inhibitor of brain GABA-T, is generally administered intracistemaliy, although high doses (2 g/kg SC) have been found to be effective [ 141. Oral ~minist~tion -. ‘Author to whom correspondence

Copyright

GAD

Enzyme turnover

would be expected to be less effective because of the instability of the compound in acidic media. This paper will describe some of our work on the mechanism of GAD and GABA-T catalysis, delineate how it was conceived that y-acetylenic GABA and y-vinyl GABA might be enzyme-activated irreversible inhibitors of GABA-T, document that indeed this is so and describe some of the in vivo characteristics of these compounds and their use in estimating the turnover of GAD and GABA-T. GABA-T is a typical pyridoxal phosphate-dependent transaminase that catalyzes the reversible transamination of GABA with the active-site pyridoxal phosphate (Py+CHO) to yield succinic semialdehyde (SSA) and the pyridoxamine (PMP) form of the enzyme-bound co-factor. The oxidized cofactor is reformed by transamination with ~-ketoglut~ate (a-KG) to yield glutamate and enzyme bound Py+CHO. The first step in the transamination reaction is the formation of a Schiff base between the y-amino group of GABA and the enzyme-bound Py’CHO, which results in activation of a proton on this y-carbon (Fig. 2). If GABA-T were to accept y-vinyl GABA or y-acetylenic GABA as a substrate analogue of GABA, then proton abstraction from the Schiff base would lead in each case to the creation of an alkylating agent in the enzyme’s active site. Figures 3 and 4 describe our proposed mechanisms for the irreversible inhibition of GABA-T by y-acetylenic GABA and y-vinyl GABA respectively. In each case inhibition could result from either of two pathways. In the first, Path a, the “suicide substrate” transaminates with the enzyme

should be addressed.

0 1980 ANKHO

International

Inc.-0361-9230/80/080375-05$01.00/O

L.IPPERT,

tiH,

N”, GABA FIG.

1(-ACETYLENIC

JUNG

AND METCALF

NH2 GABA

a-VINYL

GABA

1. Structures.

+Y Em-61

0

rx

0’

y3

-

Bow*” ,OoH * C”3 N

e

0

O

i

Y’K

0'

N\=CHPy

h-l+ In Vitro

Ii

0

0

OH

+-

FIG. 4. Proposed mechanism of inhibition of GABA-T by y-vinyl GABA; B=base in enzyme active site; Nu=nucleophilic residue in enzyme active site.

Irx

OH

N-CH,Py

FIG. 2. Schematic representation of the transamination catalyzed by GABA-T. Reproduced from [IS].

of GABA

FIG. 3. Proposed mechanism of inhibition of GABA-T by y-acetylenic GABA; B=base in enzyme active site: Nu=nucleophilic residue in enzyme active site.

yielding the acetylene or olefin conjugated to the tautomeric Schiff base. In the second, Path b, the electrons are directed into the unsatu~t~ bond yielding, after reprotona~on, the conjugated allene in the case of acetylenic GABA and the isomerized olefin in the case of vinyl GABA. Vinyl glycine inhibits aspartate aminotransferase by a mechanism analogous to this latter pathway [7], but this may be a special case: Asp-T may be able to stabilize the developing negative charge on the terminal carbon of vinylglycine, since this position corresponds to the @carboxylate of aspartate.

According to our proposed mechanisms, acetylenic and vinyl GABA should inhibit GABA-T only after the enzyme abstracts the y-proton from these substrate analogs. The inhibition should therefore be time-dependent and be subject to a primary kinetic isotope effect on the proton abstraction step. We have synthesized y-deutero-y-acetylenic GABA and y-deutero-~-vinyi GABA and have found that, indeed, there is a primary kinetic isotope effect on the rate of inhibition of GABA-T by these compounds (Fig. 5.). In the case of y-deutero-y-acetylenic GABA the effect manifests itself on the apparent Michaelis constant I&&=2.47 and not on the inactivation rate constant (Fig. 5.). Proton abstraction must therefore occur before the inhibition can occur, but proton abstraction cannot be the rate determining step in the overall inhibition process. Presumably, the rate determining step is the covalent m~~cation of the enzyme by the transformed ‘*C-y-acetylenic GABA (4-f2,3-‘4C1inhibitor. When aminohex-S-ynoic acid) is incubated with GABA-T there is a time-dependent incorporation of label into the enzyme coincident with the loss of enzymatic activity (Pig. 6). Upon complete ~n~bition 0.9 equivalents of acetylenic GABA are incorporated per J8,OOOda&on sub-unit of the enzyme. In the transamination reaction with its normal substrates GABA-T abstracts only the 4-pro-S hydrogen from GABA and the 2-S-proton from L-glutamate [2,16]. The absolute configuration of the position from which the proton is abstracted is identical in both cases (Fig. 7). Gf the two isomers of y-acetylenic GABA, it is only the 4-S isomer which, upon binding to GABA-T, could altow the same alignment of the catalytic groups as do GABA and L-glutamate. Accordingly, it is only the 4-S isomer of y-acetylenic GABA which irreversibly inhibits GABA-T [Z]; the 4-R isomer has no effect, GABA and L-glutamate, at concentrations much below their respective Km’s, effectively protect against the inhibition of GABA-T by either y-acetylenic GABA or y-vinyl GABA 19,161. This protective effect is lost in the presence of (w-KG but not in the presence of oxaloacetate 1161 (which does not t~s~inate with GABA-T). This is ~itional evidence that it is only the pyridoxd form of the enzyme that is susceptible to inhibition by y-acetylenic and y-vinyl GABA. The same enantiomer of y-acetylenic GABA (4-S-4aminohex-S-ynoic acid) as the one which inhibits GABA-T also irreversibly inhibits rn~rn~~ brain GAD 121; the 4-R isomer has no effect on this enzyme although the 4-R isomer and not the 4-S isomer inhibits bacterial GAD 1121. The 4-

REACTIONS

CATALYZED

377

BY GAD AND GABA-T

A

Ii

H*Nj./-.&COoH

n/

+i

\/VCOOH

YN’I:

*NCOOH

W’A 0

2 - S - glutamic acid

4-S-4-omino[4-‘Ifj

/I butyrk acid

4-S-4-ominohex-5-ynoic

acid

30 -

/

E ._ E

FIG. 7. Absolute configuration of substrates S-proton is abstracted in each case.

of GABA-T.

The

: *-

__

_/-

,’

,

I

I KH.72xK?Sy

1 IO

I

I

20

30

KD’It3r164M

I/I

mhl-’

20

.c E : c-

IO

0 0

I

0.2 w

2

I / I x IO’ rvl-’ FIG. 5. Double reciprocal plot of inhibition of GABA-T by: A-W----y-deutero-y-acetylenic GABA and -L-y-acetylenic GABA y-deutero-y-vinyl GABA and -H-y-vinyl B--O--GABA

6000

-

-‘W

-20

8

1,

I . 0 0

IO

20

40

60

80

TIME (minutes)

FIG. 6. Time-dependent hibition of GABA-T -0-

incorporation

of 14C-•-

by “C-y-acetylenic

during inGABA.

S-isomer is not as effective an inhibitor of mammalian GAD as of GABA-T; it has a lower affinity and a slower maximal rate of inhibition for the former than for the latter enzyme. Nevertheless, mammalian brain GAD precipitates its own inhibition by stereoselectively removing the 4-S-proton of y-acetylenic GABA, as evidenced by a primary kinetic deuterium isotope effect on the proton abstraction step [2] analogous to the one which occurs during the inhibition of GABA-T. These results suggest either that GAD can function as a transaminase on a product analogue of its normal substrate or that there is an inversion of configuration of the a-carbon of glutamate in the protonation step during GAD catalyzed formation of GABA. We have therefore investigated this problem and have determined that mammalian GAD decarboxylates L-glutamate with retention of configuration [2]. This is consistent with all previous studies of the decarboxylation of amino acids, e.g. glutamate [32], histidine [30], lysine [ 151 and tyrosine ]I] by their respective decarboxylases. The inhibition of mammalian GAD, therefore, cannot be a consequence of the microscopic reversibility principle as may be the case in the inhibition of bacterial GAD by this compound. It is consistent with a vestigial transaminase capability of mammalian GAD in line with Dunathan’s proposal [4] that all pyridoxal phosphatedependent enzymes are derived from a common ancestral enzyme. All of the above evidence, taken together, demonstrates that the rationally conceived compounds y-acetylenic and y-vinyl GABA are irreversible inhibitors of GABA-T because they are accepted as substrates by the enzyme and that each inhibitor is transformed by GABA-T to produce an alkylating agent which reacts with the enzyme before the activated inhibitor can diffuse from the enzyme’s active site. The above evidence also demonstrates that y-acetylenic GABA inhibits GAD although it reacts as neither a substrate nor product analogue. The inhibition, nevertheless, is enzyme-activated and shows that the enzyme has a hitherto undetected catalytic capability which can manifest itself when its normal catalytic reaction is not possible.

Peripheral administration (IP, IV, PO or SC) of either y-acetylenic GABA [lo] or y-vinyl GABA [ 1I] to rats or mice causes a dose-dependent, irreversible inhibition of brain GABA-T which results in increased brain GABA levels. Acetylenic GABA is more potent than vinyl GABA. This is explained, in part, by the lower dose of acetylenic GABA needed to produce an equivalent peak level of drug in the brain. For instance, within 1 hour after a dose of acetylenic GABA 100 mg/kg IP or vinyl GABA 400 mg/kg IP, peak levels in the brain are 7 pg/g and 5 pg/g respectively (P. Bohlen, unpublished results). The rapid onset of GABA-T inhibition follows the rapid penetration of the brain by either inhibitor. Since these inhibitors produce an irreversible in-

LIPPERT.

JUNG

AND METCALF

50

TIME

WTER

INJECTION

l

11.ACEYYLENIC

T

&-VINYL

( DAYS)

GABA

v

GA011

I

L

L

t

I

i

I

2

3

4

5

6

DAYS

FIG. 9. Calcufation of the turnover of GAD and GABA-T in mouse brain from the recovery of enzymatic activity following inhibition by y-acetylenic or y-vinyl GABA. The data are taken from Fig. 8 and are replotted as % inhibition on a log scale vs time.

J

TIME AFTER INJECTION ( hr) FIG. 8. Time course of effect on mouse brain GABA-T and GAD

activities and GABA levels: A, y-acetylenic GABA 100mg&g IP; B, y-vinyl GABA 1500n&kg IP. 8A was reproduced from [20]; 8B was reproduced from [ 111.

GABA, probably due to the greater effect of the former on GAD [ 10,111. After inhibition of GAD or GABA-T in vitro by acetylenic GABA, there is negligible recovery of enzymatic activity even after prolonged dialysis. The reappearance of enzymatic activity in vivo after the drug has disappeared from the brain is probably a consequence of de novo enzyme synthesis. Calculation of Turnover of GAD and GABA-T In light of the fact that brain proteins are turning over, any description of changing GAD and GABA-T levels must consider degradation as well as synthesis.

PRECURSORS k,_, hibition, the inhibition is cumulative (Fig. 8). Six hours after a single dose of acetylenic GABA (100 mglkg II?) or vinyl GABA (1500 mg/kg IP) mouse brain GABA-T is inhibited 95% and 80% respectively and brain GABA levels are increased to 5 times control levels [lO,ll]. By 12 hours after ~~inistration of acetylenic GABA the slow return of GABA-T activity and GABA levels toward control values has already begun; after administration of vinyl GABA this process does not start for at least 48 hours. The longer duration of action of vinyl GABA compared to acetylenic GABA (Fig. 8) is probably the result of the slower disappearance from the brain of the former compound. More than 1% of the peak level of vinyl GABA is still in the brain at 48 hours [ 1l] while acetylenic GABA is eliminated within 16 hours. Were it present, less than 2% of the peak level of acetylenic GABA would have been detected {P. B&len, unpublish~ results). Vinyl GABA has no effect on GAD activity in vitro [ 161 while acetylenic GABA is an irreversible inhibitor of this enzyme [2]. Nevertheless, vinyl GABA does produce a slow decrease of brain GAD activity in vivo. At any given level of GABA-T inhibition, the concentrations of GABA are lower after administration of acetylenic GABA than after vinyl

E,, k+

DEGRADED ENZYME

Assuming that a constant fraction of GAD and GABA-T are being degraded at any time [28] and that the irreversible inhibition does not affect the rate of synthesis of these enzymes [25], the rate equation governing changes in GAD and GABA-T levels can be formulated [28].

(l)

dE,

z

dt

k.....k&,=0

’

where

E,,=total enzyme protein E,=active enzyme k,=rate of synthesis kd=rate constant for degradation dE, _ k,-k&, dt therefore

and from (1) k,=kdE,,

dE,idt 1-E,E,, after integration k,=

kst B 0 but l- E,/E,,=Fractional inhibition of enzyme activity In (I-E,IE,,)=

REACTIONS

CATALYZED

379

BY GAD AND GABA-T

During the recovery process when no further inhibition is occurring there should be an exponential return of enzymatic activity [25,26]. A plot of In (% inhibition) vs time should be linear with a slope= kd = k,/E,, (Fig. 9). Furthermore, according to this scheme, the kinetics of the return of enzymatic activity are independent of the agent causing the loss of active enzyme. From the reappearance of enzymatic activity starting 1 day after the administration of acetylenic GABA we calculate a degradation rate constant of 0.29/day for GAD and 0.20lday for GABA-T (Fig. 9). Since t1h1=0.69/k, the calculated half-lives of GAD and GABA-T are 2.4 days and 3.5 days respectively. The values calculated from the recovery of enzymatic activity starting 2 days after administration of vinyl GABA (Fig. 9) are consistent with these values. Decreased brain GAD activity has been shown to result

from direct administration of GABA to chick embryos [8] or increased brain GABA levels following inhibition of GABA-T by aminoxyacetic acid [31]. It was suggested, therefore, that increased brain GABA levels act as a feedback regulator of GABA synthesis [8,31]. The slow decrease in brain GAD activity observed after administration of vinyl GABA was unexpected since no inhibitory effect was found in vitro. According to the above kinetic treatment, complete inhibition of GAD formation should result in a decline of GAD activity to 75% of control activity after 24 hours. The slow decline in GAD activity is therefore consistent with an inhibition of de note synthesis of GAD induced by the sustained high GABA levels resulting from the administration of vinyl GABA. The exact mechanism by which GAD is decreased is currently under investigation.

REFERENCES 1. Belleau, B. and J. Burba. The stereochemistry of the enzymic decarboxylation of amino acids. J. Am. Chem. Sot. 82: .57515752, 1960. 2. Bouclier, M., M. J. Jung and B. Lippert. Stereochemistry of reactions catalyzed by mammalian-brain L-glutamate I-carboxy-lyase and 4-aminobutyrate: 2-oxoglutarate aminotransferase. Eur. J. Biochem. 98: 363-368, 1979. 3. DaVanzo, J. P., R. J. Mathews, G. A. Young and F. Wingerson. Studies on the mechanism of action of aminooxyacetic acid. Toxic appl. Pharmac. 6: 396-401, 1964. 4. Dunathan, H. C. and J. G. Voet. Stereochemical evidence for the evolution of pyridoxal-phosphate enzymes of various function from a common ancestor. Proc. nafn. Acad. Sci. U.S.A. 71: 3888-3891, 1974. 5. Fowler, L. J. and R. A. John. Active-site-directed irreversible inhibition of rat brain 4-aminobutyrate aminotransferase by ethanolamine-O-sulphate in vitro and in viva. Biochem. J. 130: 56%573, 1972. 6. Fowler, L. J. Analysis of the major amino acids of rat brain after in viva inhibition- of GABA iransaminase by ethanolamine 0-sulvhate. J. Neurochem. 31: 437-440, 1973. 7. Geh&g, H., R. Rando and P. Christen. Active-site labelling of aspartate aminotransferases by the P-y-unsaturated amino acid Vinylglycine. Biochemistry 16: 4832-4836, 1977. 8. Haber, B., P. Y. Sze, K. Kuriyama and E. Roberts. GABA as a repressor of L-glutamic acid decarboxylase in developing chick embryo optic lobes. Brain Res. 18: 545-547, 1970. 9. Jung, M. J. and B. W. Metcalf. Catalytic inhibition of y-aminobutyric acid-a-ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog. Biochem. hiophys. Res. Commun. 67: 301-306, 1975. 10. Jung, M. J., B. Lippert, B. W. Metcalf, P. J. Schechter, P. Biihlen and A. Sioerdsma. The effect of 4.aminohex-5-vnoic acid (y-acetylenic‘GABA, y-ethynyl GABA) a catalytic inhIbitor of GABA transaminase on brain GABA metabolism in viva. J. Nrurochem. 28: 717-723, 1977. 11. Jung, M. J., B. Lippert, B. W. Metcalf, P. Biihlen and P. J. Schechter. y-Vinyl GABA (4-aminohex-5-enoic acid) a new selective irreversible inhibitor of GABA-T: Effects on brain GABA metabolism in mice. J. Neurochem. 29: 797-802, 1977. 12. Jung, M. J.. B. W. Metcalf, B. Lippert and P. Casara. Mechanism of the stereospecific irreversible inhibition of bacterial glutamic acid decarboxylase by (R)-(-)-4-aminohex-5-ynoic acid, an analog of 4-aminobutyric acid. Biochemisfry 17: 26282632, 1978. 13. Krjewic, K. Chemical nature of synaptic transmission in vertebrates. Physiol. Rev. 54: 418-540, 1974. 14. Leach, M. J. and J. M. G. Walker. Effect of ethanolamine-Osulphate on regional GABA metabolism in mouse brain. B&hem. Pharmac. 26: 1569-1572, 1977. 15. Leistner, E. and I. D. Spenser. Stereochemistry of the enzymic decarboxylation of L-lysine. J. Chem. Sot. Chem. Commun. 378-379, 1975. 16. Lippert, B., B. W. Metcalf, M. J. Jung and P. Casara. 4-Aminohex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyrate aminotransferase in mammalian brain. Eur. J. Biochem. 74: 441-445, 1977.

17. Lloyd, K. G. and 0. Hornykiewicz. L-glutamic acid decarboxylase in Parkinson’s disease: Effect of L-dopa therapy. Nnture 243: 521-523, 1973. 18. Meldrum, B. S. Epiliepsy and y-aminobutyric acid mediated inhibition. Int. Rev. Nc>urohio/. 17: l-36, 1975. 19. Metcalf, B. W. Inhibitors of GABA metabolism. Biwhem. Phtrrmtrc. 28: 1705-1712, 1979. 20. Metcalf, B. W., M. J. Jung, B. Lippert, P. Casara, P. Biihlen and P. J. Schechter. In: GABA-Neurofransmitfers, Alfred Benzon Symposium 12, edited by P. Krogsgaard-Larsen, J. Scheel-Kriiger and H. Kofod. Covenhaaen: Munksaaard. 1978. _ pp. 2X-246: 21. Metcalf, B. W. and P. Casara. Regiospecific 1,4 addition of a propargylic anion. A general synthon for 2-substituted propargylamines as potential catalytic irreversible enzyme inhibitors: Tetrahedron Lett. 3337-3340, 1975. basis for the irrever22. Metcalf. B. W. and M. J. Jung. Molecular sible inhibition of 4-aminobityric acid: 2-oxoglutarate and L-omithine: 2-oxoacid aminotransferases by 3-amino-1,5cvclohexadienvl carboxylic acid (isogabaculine). Molec. Pharn& 16: 53%j45, 1979.. 23. Palfrevman. M., S. Huot, B. Livvert . _ and P. J. Schechter. The effect bf y-acetylenic GABA, an enzyme-activated irreversible inhibitor of GABA-transaminase, on dopamine pathways on the extrapyramidal and limbic systems. Eur. J. Phcrrmcrc. 50: 325336, 1978. 24. Perry, T. L., S. Hansen and M. Kloster. Huntington’s chorea: deficiency of y-aminobutyric acid in brain. Nr,~a Engl. J. Med. 288: 337-342, 1973. R. Hartley, Jr. and M. 25. Price, V., W. Sterling, V. Tarantola, Rechcige, Jr. The kinetics of catalase synthesis and destruction in l?vo. J. hiol. Chem. 237: 3468-3475, 1962. by nitro26. Quiring, K. and D. Palm. Inhibition of amineoxidases furan derivatives: Possible structure-activity relations and criteria for irreversibility. Naunyn-S~,hmiedehrrKs Arch. Pharmtrc’. 265: 397410, 1970. 27. Roberts, E. In: GABA in Nenwus System Fttnc~tiou, edited by E. Roberts, T. Chase and D. Tower. New York: Raven Press, 1976, pp. 515-539. R. Control of enzyme levels in mammalian tissues. 28. Schimke, Adv. Enzymol. 37: 135-187, 1973. M. Maitre, H. Randrianarisoa and P. 29. Simler, S., L. Ciesielski, Mandel. Effect of sodium-n-dipropylacetate on audiogenic seizures and brain y-aminobutyric acid level. Biochem. Pharmac,. 22: 1701-1708, 1973. of Lac30. Snell, E. E. and G. W. Chang. Histidine decarboxylase tobacillus 30a. II. Purification, substrate specificity, stereospecificity. Biochemistr?: 7: 2005-2012, 1968. 31. Sze, P. Y. and R. A. Lovell. Reduction of level of I-glutamic acid decarboxylase by y-aminobutyric acid in mouse brain. J. Neurochem. 17: 1657-1664, 1970. 32. Yamada, H. and M. O’Leary. Stereochemistry of reactions catalyzed by glutamate decarboxylase. Biochemists 17: 669671, 1978. I

_