Kinetic Studies on the Inhibition of GABA-T by g-VinylGABA and Taurine

SABA A.J. SULAIMANa,*, FAKHR ELDIN O. SULIMANa,† and SAMIRA BARGHOUTHIb

aSultan Qaboos University, Department of Chemistry, College of Science, Box 36, Al-Khod 123, Sultanate of Oman; bArab American University atJenin, Department of Chemistry, College of Arts and Sciences, Box 240, Jenin, Palestine

(Received 18 October 2002; In final form 7 March 2003)

g-Aminobutyric acid transaminase (GABA-T, EC 2.6.1.19)is a pyridoxal phosphate (PLP) dependent enzyme thatcatalyzes the degradation of g-aminobutyric acid. Thekinetics of this reaction are studied in vitro, both in theabsence, and in the presence of two inhibitors: g-vinylGABA (4-aminohex-5-enoic acid), and a natural product,taurine (ethylamine-2-sulfonic acid). A kinetic model thatdescribes the transamination process is proposed. GABA-Tfrom Pseudomonas fluorescens is inhibited by g-vinylGABA and taurine at concentrations of 51.0 and 78.5 mM.Both inhibitors show competitive inhibition behaviorwhen GABA is the substrate and the inhibition constant(Ki) values for g-vinyl GABA and taurine were found to be26 6 3 mM and 68 6 7 mM respectively. The transamin-ation process of a-ketoglutarate was not affected by thepresence of g-vinyl GABA, whereas, taurine was anoncompetitive inhibitor of GABA-T when a-ketogluta-rate was the substrate. The inhibition dissociation constant(Kii) for this system was found to be 96 6 10 mM: TheMichaelis-Menten constant (Km) in the absence ofinhibition, was found to be 0:79 6 0:11 mM; and 0:47 60:10 mM for GABA and a-ketoglutarate respectively.

Keywords: g-Aminobutyric acid transaminase (GABA-T); g-VinylGABA (4-aminohex-5-enoic acid); Taurine (ethylamine-2-sulfonicacid); Kinetics; Inhibitors

Abbreviations: GABA, g-Aminobutyric acid; GABA-T, g-Amino-butyric acid transaminase; SSDH, succinic semialdehyde dehy-drogenase; GAD, glutamic acid decarboxylase; PLP, pyridoxalphosphate

INTRODUCTION

g-Aminobutyric acid (GABA) is a major inhibitoryneurotransmitter in the mammalian central nervous

system. It is the most prevalent and abundantneurotransmitter in the brain with as many asone-third of all neurons in the brain using GABA asan inhibitory neurotransmitter.1

The adequate control of GABA levels in the brain isattributed to the regulation of the levels of the anabolicenzyme, glutamic acid decarboxylase (GAD; EC4.1.1.15), and the catabolic enzyme, g-aminobutyricacid transaminase (GABA-T; EC 2.6.1.19).2 This can berepresented by the following equations:

L–Glutamic AcidGAD

������! GABA þ CO2 ð1Þ

GABA þ a2 KetoglutarateGABA–T������! Succinic

Semialdehyde þ Glutamate ð2Þ

It is well known that reaction (2) undergoes Schiffbase formation at the active site of GABA-T that usespyridoxal-5-phosphate (PLP) as a co-enzyme.3

In disorders such as epilepsy, Huntington’schorea, and schizophernia, GABA levels are detectedto be lower than normal. Here, inhibition of GABA-Tbecomes necessary to increase the levels of GABAand to bring them back to normal.4

Inhibition of GABA-T can be accomplished by anumber of different inhibitors that vary in theirinhibitory effect towards GABA-T. Examples ofstructural analogue inhibitors include amino oxo-acetic acid (AOAA),5 sodium-n-dipropylacetate(n-DPA),3 and g-vinyl GABA. In fact, over the lastthree decades, there have been several studiessuggesting g-vinyl GABA, known as vigabatrin, asa potent inhibitor of GABA-T in vivo and in vitro.6,7,4,8

ISSN 1475-6366 print/ISSN 1475-6374 online q 2003 Taylor & Francis Ltd

DOI: 10.1080/1475636031000118428

*Current address: Higher College of Technology, P.O Box 74, PC 133, Muscat, Oman.†Corresponding author. Fax: þ968-515469E-mail: fsuliman@squ.edu.om

Journal of Enzyme Inhibition and Medicinal Chemistry, August 2003 Vol. 18 (4), pp. 297–301

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Taurine, on the other hand, is a natural productfound in the central nervous system, skeletal muscles,and is very concentrated in the heart and brain.9

Taurine is a sulfur containing amino acid which isavailable in the diet but can be synthesized in the bodyfrom cysteine. It is also obtained as a cleavage productof taurcholic acid (the sodium salt of this acid occurs inthe bile). Nevertheless, the functional role of taurine inthe body remains unknown. There are reports thatdescribe taurine as an inhibitory neurotransmitter inthe brain.10,11 Other workers prefer to consider taurineas modulator rather than a neurotransmitter.12,13 Therehave been reports on the benefits of taurine supple-mentation for epileptics, membrane stabilization forthe muscles and nerves, and in the prevention of heartfailure. In this work, we study the kinetics of inhibitionof GABA-T using two inhibitors, g-vinyl GABA andtaurine, in an attempt to comparatively assess theirinhibitory effect towards this enzymatic system.

METHODOLOGY

Materials and Methods

Enzymes, Substrates, and Co-Factors. GABase fromPseudomonas fluorescens was purchased from Sigma-Aldrich Chemical Co (Germany). The cell freepreparation contains both g-aminobutyric acid trans-aminase (GABA-T) and succinic semialdehyde dehy-drogenase SSDH (EC 1.2.1.24) with 44% protein(biuret) and total activity of 0.995 units/mg protein.One unit of GABase converts one mmole ofg-aminobutyric acid to succinic semialdehyde andthen to succinate per minute with a stoichiometricreduction of one mmole of NADPþ at pH 8.6 and 258C.The chemicals GABA, sodium salt of a-ketoglutarate,NADPþ, potassium pyrophosphate, potassium phos-phate, and 2-mercaptoethanol were reagent gradefrom Sigma-Aldrich Chemical Co. g-Vinyl GABA(Vigabatrin) was purchased from Marion MerrellDow Co, and mega taurine was purchased from TwinLaboratories Inc. (Newyork, U.S.A).

Initial Velocity Measurements

GABA-T activity was measured by a coupledspectrophotometric assay using a Diode Arrayspectrophotometer HP 8453. In this procedureGABA is converted into succinic semialdehyde byGABA-T and then to succinate by succinic semi-aldehyde dehydrogenase (SSDH) with a stoichio-metric reduction of NADPþ to NADPH according tothe following equation.

Succinic Semialdehyde

þ NADPþ SSDH������! Succinate þ NADPH ð3Þ

The redox reaction was followed spectrophotome-trically by recording the rate of increase in absorptionat 340 nm at a temperature of 258C. Reaction mixtureswere made up to 3.00-ml containing potassiumpyrophosphate (0.1 M) pH 8.6, GABA (0.2–7.0 mM),a-ketoglutarate (0.09–5.0 mM), NADPþ (1.66 mM),and inhibitor (51.0 and 78.5 mM) for the kinetic studiesin presence of inhibitor. The reaction was initiated byadding 0.02 ml of the standardized enzyme solution.Initial velocities were based upon the early linearportions of the reaction curve. The kinetic studies wereperformed by following the time course for GABA-Tcatalyzed transamination of GABA at different con-centrations of a-ketoglutarate (4.0, 6.8 and 10.0 mM) ata pH of 8.6 and a fixed temperature of 258C. Initialvelocities were obtained on the basis of the initial linearportion of the time courses for the reactions.

Data Analysis

Kinetic parameters were obtained by nonlinearregression analysis using the programs EZ-Fitecurve fitter14 and Enzfitter [Biosoft]. The substratesaturation curves of a Ping-Pong mechanism werefitted to equation (4). Data conforming to competi-tive and non-competitive inhibition were fitted toequations (5) and (6) respectively.

n ¼Vmax½A�½B�

ðKA½B� þ KB½A� þ ½A�½B�Þð4Þ

n ¼Vmax½A�

ðKAð1 þ ½I�=KiÞ þ ½A�Þð5Þ

n ¼Vmax½B�

ðKBð1 þ ½I�=KiÞ þ ½B�ð1 þ ½I�=KiiÞÞð6Þ

where n is the initial velocity, Vmax is the maximumvelocity. A and B are the substrates GABA, anda-ketoglutarate respectively. KA and KB are Km

values for the substrates A and B respectively; Ki andKii are the inhibition dissociation constants for theinhibitor (I). EZ-fit uses two nonlinear methods,Gauss–Newton method, and the simplex method ofNelder and Mead.15 In all cases the best modelselected is always the simplest one that passes allstatistical tests; the one that has the lowest x2 andbest correlation coefficient.

RESULTS AND DISCUSSION

g-Aminobutyric acid transaminase (GABA-T) is theenzymatic system that catalyzes the reaction:

GABA þ a2 KetoglutarateGABA–T�����������!

Succinic Semialdehyde þ Glutamateð7Þ

The enzyme (GABA-T) is tightly bound to theco-enzyme pyridoxal phosphate. This holo-enzyme

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transaminates with each substrate in turn, thusinvolving two forms of the holo-enzyme; the aldo-form (EZCHO), and amino form (EZCH2NH2).3

Therefore, the above reaction can be convenientlydivided into two half-reactions:

GABA þ EZCHO O Succinic Semialdehyde

þ EZCH2NH2 ð8Þ

EZCH2NH2 þ a2 Ketoglutarate O Glutamate

þ EZCHO ð9Þ

Figure 1 shows a double reciprocal plot in thedirection of GABA transamination. A group ofparallel lines is obtained conforming with a Ping-pong mechanism. The Km values for each substratewere determined using Equation (4) and the kineticparameters obtained are summarized in Table I.The Michaelis-Menten constants obtained arecomparable to Km values reported for GABA-Tfrom Pseudomonas fluorescens.3,16

Inhibition Studies

Two structural analogues of the substrate weretested for their ability to inhibit GABA-T: g-vinylGABA and taurine (Scheme 1). g-Vinyl GABA is adrug used by patients suffering from epilepsy andschizophrenia whereas the latter is a natural productthat has proved useful to those patients. Few studieshave been performed on the kinetics and themechanism of action of taurine.

The steady state assays in the presence of g-vinylGABA demonstrated competitive inhibition withrespect to GABA as shown in Figure 2. The inhibitiondissociation constant, Ki, for this inhibitor was foundby fitting the data to Equation (5) where a value of26 ^ 3 mM was obtained. Competitive inhibitionbehaviour indicates the affinity of the inhibitor forthe substrate-binding site. The different degree of

FIGURE 1 Initial velocity patterns of GABA transamination atvarying concentration of a-ketoglutarate concentration: (O)4.0 mM, (B) 6.8 mM, (X) 10.0 mM. pH 8.6, temperature 258C.Points are experimental and solid lines are from a fit usingEquation (4).

FIGURE 2 Inhibition by g-vinyl GABA with respect to GABA.Assays were carried out at pH 8.6, ½a2 ketoglutarate� ¼ 6:8 mMand temperature 258C: g-vinyl-GABA ¼ (†) 0 mM, (B) 51.0 mM,(O) 79.0 mM. Points are experimental and solid lines are from a fitusing Equation (5).

SCHEME 1 Structures of g-vinyl GABA and taurine.

TABLE I Kinetic parameters for GABA transamination byGABA-T in the absence and presence of the inhibitors g-vinyl-GABA and taurine

Parameter GABA a-Ketoglutarate

Vmax 0.91 ^ 0.03 mMKm 0.79 ^ 0.11 mM 0.47 ^ 0.10 mMKi (g-vinyl-GABA) 26 ^ 3 mM -Ki (Taurine) 68 ^ 7 mMKii (g-vinyl-GABA) - -Kii Taurine) 96 ^ 10 mM

KINETICS OF INHIBITION OF GABA-T 299

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inhibition can be explained by the similaritiesbetween the substrate and its analogues.

Interestingly, when a-ketoglutarate is varied atfixed concentrations of GABA, g-vinyl GABA had noeffect on the initial rates. This result indicates clearlythat g-vinyl GABA has no inhibitory effect on theamino-form of the enzyme. This agrees withpublished information such as the studies onincubation of rat brain GABA-T with g-vinyl GABAthat was found to decrease the enzymatic activity.17

Other studies showed that inhibition in vivo followspseudo first order kinetics and was described asbeing in accord with a ping-pong bi bi mechanism.

The reciprocal plots of varied GABA concentration(a-ketoglutarate concentration is fixed at 6.8 mM) inthe presence of different taurine concentrationsdemonstrated a similar effect on GABA-T to that ofg-vinyl GABA (Figure 3). The patterns in Figure 3provide clear evidence of a competitive inhibition.However in this case taurine is a weaker inhibitorwith respect to GABA with a Ki value of 68 ^ 7 mMcompared to a Ki of 26 ^ 3 mM for g-vinyl GABA.Competitive inhibition indicates that taurine andg-vinyl GABA can bind to the same form of theenzyme, which is the aldehyde form. By comparisong-vinyl GABA and taurine are similar to GABA withrespect to the amino part of the molecule, andtherefore expected to bind in a similar fashion to thesame enzyme site. However, it is clear thatthe presence of the ZSO2

3 group in taurine in placeof the ZCH2ZCOOH group resulted in the poorerbinding of taurine. This could be due to the fact thatthe structural differences result in more stableintermediates or the enzyme achieves a high degree

of specificity that favours certain configurations ofthe substrates. The mechanism by which g-vinylGABA and taurine inhibit GABA-T could be similarto the well-known mechanism for the inhibition ofGABA-T by g-ethynyl-GABA.18 In this mechanisman aldimine intermediate is formed between theinhibitor and the pyridoxal-50-phosphate that mightbe stabilized internally by hydrogen bonding. Lossof an a-hydrogen produces a resonance-stabilizedspecies by electron pair delocalization. This species isexpected to be more stable for g-vinyl-GABA thanfor taurine. This is then followed by the tautomeriza-tion of the imine to form another intermediate whichupon attack of an enzyme active-site nucleophilewould give a covalently inactive enzyme. Anotherpossible mechanism involves formation of an iminebetween the coenzyme and the inhibitor followed bya step in which the enzyme catalyzes the isomeriza-tion of the intermediate into a reactive conjugateolefin.19 Nucleophilic addition by an active sitenucleophile would result in the formation of acovalently modified enzyme.

Taurine was also found to inhibit GABA-T in itsamino form when GABA concentration was fixedand a-ketoglutarate concentration was varied.Figure 4 represents a double reciprocal plot of theinitial velocity vs. a-ketoglutarate concentration andshows that taurine demonstrates a non-competitivepattern of inhibition with respect to the amino formof the enzyme. The value of the inhibitiondissociation constant Kii for taurine is 96 ^ 10 mMand is obtained by fitting the initial velocity data toEquation (6). In this type of inhibition a decrease inthe amount of the active enzyme would probably

FIGURE 3 Inhibition by taurine with respect to GABA. Assayswere carried out at pH 8.6 and temperature 258C: taurine ¼ (X)0 mM, (B) 51.0 mM, (O) 79.0 mM. Points are experimental andsolid lines are from a fit using Equation (5).

FIGURE 4 Inhibition by taurine with respect to a-ketoglutarate.Assays were carried out at pH 8.6 and temperature 258C: taurine¼ (X) 0 mM, (O) 51.0 mM, (B) 79.0 mM. Points are experimentaland solid lines are from a fit using Equation (6).

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result from the formation of an enzyme-substrate-inhibitor complex. This reaction decreases theproportion of the central complexes that proceed toproducts, and as a result non-competitive inhibitionis observed.

In this study the slight upward curvature in thedouble reciprocal plots of the initial velocity ofGABA (Figure 1) is a clear indication of substrateinhibition. Furthermore when the data in Figure 1were subjected to a fit involving substrate inhibition,slight improvement in the results was obtained butwithout a significant change in the values of theMichaelis-Menten constants. Similar findings werereported in the literature, as substrate inhibition isvery common in ping-pong mechanisms.3,13 Usuallysubstrate inhibitions may result from the interactionof the substrate with improper forms of the enzymethat would form dead-end complexes. In generaldead-end complexes may result from two substratescompeting for a single enzyme-binding site thatexists in two forms, or one substrate binding at twosites in a two-site enzyme.

It is worth mentioning here that the enzymaticmechanism and the inhibition patterns of GABA-Tfrom Pseudomonas fluorescens and from mammaliansources obey a ping-pong bi bi reaction mechan-ism. It is therefore imperative to indicate thatGABA-T from a bacterial source is similar to acertain extent to that from the mammalian source.However, reported elsewhere, Km values forGABA and a-ketoglutarate were significantlylower for the mammalian brain GABA-T compared

to the bacterial enzyme. This may be due tothe existence of possible specific isozymes ofGABA-T.3,20

References

[1] Siegel, G.J., Agranoff, B.W., Albers, W.R. and Molinoff, P.B.(1993) Basic Neurochemistry, 5th Ed. (Raven Press).

[2] Jung, M.J., Lippert, B., Metcalf, B.W., Schechther,P.J., Bohlen, P. and Sjoerdsma, A. (1977) J. Neurochem. 28,717.

[3] Porcelli, F., Scibona, G., Lorenti, G., Mazzei, F. and Botre, C.(1996) Ber. Bunsenges. Phys. Chem. 100, 671.

[4] Larsson, O.M., Gram, L., Schousboe, I. and Schousboe, A.(1985) Neuropharmacol. 25, 617.

[5] Cassel, G., Karlsson, L. and Sellstrom, A. (1991) Pharmacol.Toxicol. 69, 238.

[6] Lippert, B., Metcalf, B.W., Jung, M.J. and Casara, P. (1977)J. Biochemistry 14, 441.

[7] Jung, M.J., Lippert, B., Metcalf, B.W., Bohlen, P. and Schechter,P.J. (1977) Tetrahedron Lett. 38, 3337.

[8] Engelborghs, S., Pickut, B.A., D’Hooge, R., Wiechert, P.,Haegele, K. and De Deyn, P.P. (1998) Drug Res. 48, 713.

[9] www.darkdog/.com/whatistaurine[10] www.pcuf.fi/jmettala/poster[11] www.tex-is.net/users/keking/taurine[12] Kruk, Z.L. and Pycock, C.J. (1991) Neorotransmitters and Drugs,

3rd Ed. (Chapmann and Hall, New York).[13] Graces, E. and Cleland, W.W. (1969) Biochemistry 8, 633.[14] Perrella F.W., Http://www.jlc.net/fperrell/webps03.htm[15] Nelder, J.A. and Mead, R. (1965) Comput. J. 7, 308.[16] Scott, E.M. and Jakoby, W.B. (1959) J. Biol. Chem 234, 932.[17] Lippert, B., Jung, M.J. and Metcalf, B.W. (1980) GABA

Neurotransmission Brain Res. Bull. 5, 375–379.[18] Burke, R.B. and Silverman, R.B. (1991) J. Am. Chem. Soc. 113,

9329.[19] Fowler, L.J., Beckford, J. and John, R.A. (1975) Biochem.

Pharmacol. 24, 1267.[20] Sherif, F.M. and Ahmed, S.S. (1995) Clin. Biochem. 28, 145.

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