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A Study of the Enzyme GABA-Transaminase to Design Inhibitory Drugs for the Treatment of Epilepsy

A Study of the Enzyme GABA-Transaminase to Design Inhibitory Drugs for the Treatment of Epilepsy
Epilepsy is a neurological condition classified as a seizure disorder.  The disorder was described as early as 400 B.C. by the Greek physician Hippocrates in a book he wrote on the "sacred" disease.  He argued that this disease was not a curse but a brain disorder that had a natural cause just like any other disease. Epilepsy is also described in the book of Mark in the Bible when Jesus is asked to cast a demon out of a small boy.  The description of the boy's actions leads to the conclusion that it is epilepsy and not a demon that was afflicting him.1   Therefore, epilepsy is a disorder that has been affecting the lives of humans and animals for hundreds of years.  Today, it is a common disorder that affects approximately 50 million people around the world.2,3  The primary effects of epilepsy are exerted on the central nervous system and are the result of a disturbance in the electrical activity of the brain's neurons.  The cause of this disturbance is believed to be an upset in the levels of excitatory and inhibitory neurotransmitters in the brain.2,3    This paper will focus on the role of the inhibitory neurotransmitter, g-aminobutyrate (GABA).  Low levels of GABA in the brain are known to result in epilepsy and other neurological disorders including Huntington's disease and Parkinson's disease.4,5  The neurotransmitter upset is normally localized in areas of the brain that have been damaged but is capable of spreading to other brain areas or being genetically inherited in certain types of epilepsy.  This chemical imbalance can be caused by a variety of factors that ultimately result in the main characteristic of epilepsy, a seizure.  While scientists know certain circumstances are capable of causing seizures, most seizures cannot be linked to any specific cause.  Depending on the type of seizure experienced, epilepsy can have an effect on movement, emotions, sensations, and may cause a person to lose consciousness.2   No matter what type of seizure is experienced it is clear there is a decrease in the person's ability to function normally on a day-to-day basis.  The myriad symptoms and the facts regarding the number of people around the world impacted by epilepsy necessitate ongoing research and treatment development.

Today the most common treatment method is with antiepileptic drugs.3   Since the development of phenobarbitol, the oldest antiepileptic drug still in use, there have been many new antiepileptic drugs developed, some of which having been more successful than others.1   Vigabatrin is one of the newly developed antiepileptic drugs used to treat seizures that are not capable of being controlled by other drugs.  Its method of action is the inhibition of g-aminobutyrate transaminase (GABA-T).3  By blocking the action of this enzyme, Vigabatrin is able to raise the levels of GABA in the brain and terminate the epileptic event.  Although Vigabatrin elevates GABA levels in the brain of a number of individuals, it only prevents seizures in 2% of patients with refractory epilepsy.  In addition to the low success rate, patients have also reported numerous side effects.3   The combination of these two factors is evidence for the requirement of a new antiepileptic drug that is more efficient at terminating the epileptic events and allowing patients live a better quality life.
The development of a new drug requires extensive knowledge concerning the mechanism of action the drug is going to take.  Antiepileptic drugs can inhibit a variety of pathways, all of which lead to an increased level of GABA in the brain.  The mechanism focused on in this paper is GABA-T inhibition.  GABA-T is a pyridoxal-5'-phosphate (PLP) dependent enzyme responsible for the catabolism of GABA into succinic semialdehyde.4,5  Therefore, preventing GABA-T from catalyzing its normal reaction will keep the levels of GABA in the brain from falling.  In order to develop a GABA-T inhibitory drug, researchers must study the structure of the enzyme and determine how it catalyzes the conversion of GABA into succinic semialdehyde.  Extensive research has already been done on several mammalian forms of GABA-T, including bovine and porcine GABA-T.  This paper is primarily focused on research related to human GABA-T, which uses the information from past mammalian GABA-T studies as a guideline.

Past studies have shown mammalian GABA-T to be a homodimeric protein.  Each subunit is 50 kDa, and there is one molecule of PLP bound per dimer.4,5  In porcine GABA-T, PLP is known to form a Schiff base with the å-amino group of lysine (Lys) 330.  When the molecule of PLP is bound to the enzyme, the absorption spectrum shows peaks at 330 and 415 nm, corresponding to a phospho-pyridoxyl chromophore and a Schiff base, respectively.  The Schiff base is the one that is formed between the Lys 330 residue on GABA-T and PLP (Lys).4  The dependence of GABA-T on PLP and the fact that PLP binds via Lys 330 make this residue critical for the catalytic activity of the enzyme. 
Studies have also indicated the two monomers of GABA-T to be connected by a disulfide bond between two cysteine (Cys) residues.5   This makes at least one Cys per monomer required for the catalytic activity because the dimer is the active form.  The GABA-T enzyme present in porcine brains is capable of being inactivated by 5, 5'-dithiobis-2-nitrobenzoic acid (DTNB).5  DTNB is a chemical that reacts with sulfhydryl groups to form a mixed disulfide bond.  This means that if DTNB reacts with a sulfhydryl group that is normally involved in a disulfide bond, it will prevent that disulfide bond from forming.  When this reaction is complete, the chromophore 5-mercapto-2-nitrobenzoic acid is liberated.  The formation of this mixed disulfide bond is reversible by the addition of 2-mercaptoethanol to the solution.  In porcine GABA-T, DTNB was discovered to react with 1.2 sulfhydryl groups and lead to the inactivation of the enzyme.5    This information guided researchers when they began studying human GABA-T.

Research shifted to human GABA-T when studies using monoclonal antibodies against bovine GABA-T indicated that it is immunologically distinct from human GABA-T, despite the fact that their amino acid sequences are more than 90 % homologous. This means that the active site for the human GABA-T could have a different environment than bovine GABA-T, therefore behaving differently when drugs based on bovine GABA-T interact with it. 4,5   This could cause the drugs to be more or less effective and could cause them to be toxic.  Research has shifted to human GABA-T in hopes of developing new antiepileptic drugs that will be more specific in their mechanisms and avoid complications. 

Based on the knowledge that Lys and Cys are required for catalytic activity in other mammalian GABA-Ts, researchers used polymerase chain reaction (PCR)-based site-directed mutagenesis to mutate these two amino acid residues in human GABA-T and then observe the effect on the enzyme's activity and its structure.  Each amino acid was mutated separately, and several mutations were introduced.  Lys-357 was mutated to asparagine, aspartic acid, glutamine, and alanine.4   Cys-321 was mutated to methionine, serine, alanine, glycine, lysine, and aspartic acid.5   The same set of procedures was used for both Lys and Cys to introduce the mutations, induce protein production, purify the proteins, and determine the catalytic activity.  PCR-based site-directed mutagenesis was used to introduce the desired mutations into the human GABA-T gene.  The PCR products were purified using agarose gel electrophoresis.  Both mutant and wild type GABA-T sequences were ligated into pGEX 4T-1 expression vector and transformed into E. coli JM109 cells.  Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to the cells to induce protein production.

Once the cells were grown, a centrifugation was performed to harvest the cells and the resulting supernatant was purified.  The purification technique used a bulk GST purification system.4,5  This system ligates the DNA encoding a glutathione-S-transferase (GST) domain with the DNA for GABA-T.  The result is a GST domain fused to the GABA-T enzyme.  This is a very efficient method for protein purification, because GST binds glutathione with an extremely strong affinity.  The column used contains glutathione-Sepharose beads, so when the protein moves through the column, the GST domain will bind tightly to the beads and be separated from the rest of the supernatant.  The protein can then be eluted from the column and the GST moiety can be cleaved using thrombin.6  The solution that results can be purified using ion exchange chromatography. 

The result of protein purification is GABA-T, which was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).  SDS-PAGE gels for both Lys and Cys experiments gave bands of 50 kDa, corresponding to the monomer of GABA-T.  The identity of the purified protein was confirmed using monoclonal antibodies against human GABA-T.  Protein concentration was determined by performing a Bio-Rad assay, and enzymatic activity was measured using an activity assay.  The activity assay measured the rate at which GABA was converted into succinic semialdehyde, by monitoring the absorbance at 340 nm for 2 min.4,5   The results of this assay and the Bio-Rad assay were used to calculate the specific activities of both the wild type and mutant GABA-T enzymes.  In the case of both experiments, the wild type GABA-T had a specific activity of 17.5 mmol·min-1·mg-1, which is close to the specific activity of bovine GABA-T (18.0 mmol·min-1·mg-1).4,5  The high specific activity value shows that the enzymes were catalytically active.  All of the mutant GABA-Ts from both the Lys and Cys experiments possessed specific activities of 0.001 mmol·min-1·mg-1.4,5  The low value shows that they had little to no catalytic activity as a result of the mutations they possessed. 

In the experiment where Lys mutations were introduced, the researchers measured the absorbance spectrum of both wild type and mutant GABA-T.  The wild type GABA-T showed the same absorption spectrum as the other mammalian forms with absorbance peaks at 330 and 415 nm, indicating wild type human GABA-T possesses a phospho-pyridoxyl chromophore and a Schiff base.  The absorption spectrum of the mutant GABA-T had no absorption peaks at either value, indicating that PLP was not capable of binding to the mutant enzymes. 

To further examine the relationship between GABA-T and PLP, apoenzymes of GABA-T were prepared.  To accomplish this, the holoenzyme was subjected to conditions to cause the PLP molecule to dissociate from the enzyme.  The resulting solution was dialyzed to remove the unbound PLP.  The catalytic activity of the resulting apoenzyme was measured using the same enzyme assay as before.  The catalytic activity of the wild type GABA-T apoenzyme was 0.00 mmol·min-1·mg-1, indicating that without PLP bound, GABA-T is completely inactive.  When PLP was added back to the apoenzyme solution, the catalytic activity was measured and found to be 16.8 mmol·min-1·mg-1, signifying 95% of the activity returned.  When PLP was added to the mutant GABA-T there was no increase in catalytic activity.  The mutant enzyme's lack of catalytic ability is the result of the missing Lys-357 residue.  Lys residues 60, 191, 318 and 470 were also mutated but did not cause the catalytic activity of GABA-T to significantly decrease, suggesting that these residues are not involved in catalysis. All of these results point to the fact that Lys-357 in human GABA-T is required for the cofactor PLP to bind to the active site.  When PLP binds, it is thought to instigate conformational changes within GABA-T.  Only after PLP has bound and these conformational changes occur can the substrate bind correctly to the active site and the reaction take place.

In other PLP-dependent enzymes, the cofactor molecule is known to form a Schiff base with the e-amino group of a Lys residue that is conserved at the active site.  While the results of this experiment suggest that Lys-357 is the conserved residue in GABA-T to which PLP binds, it cannot be confirmed until a three-dimensional structure analysis of the active site has been done.  However, it can be confirmed that Lys-357 is involved in at least helping the cofactor bind and is required for catalytic activity of GABA-T.4

In the second experiment Cys mutations were introduced.  Wild type and mutant GABA-Ts were reacted with DTNB.  The number of sulfhydryl groups modified in this reaction was measured by monitoring the release of 2-nitro-5-mercaptobenzoate at 412 nm. It was found that 1.5 sulfhydryl groups per dimer reacted in wild type GABA-T.  The reaction of these 1.5 sulfhydryl groups resulted in a 95% loss of GABA-Ts catalytic activity.  The fact that human GABA-T reacted with 1.5 sulfhydryl groups and porcine GABA-T with 1.2 can be attributed to the fact that the two enzymes are immunologically distinct.  As a result of this immunological difference the active sites are somewhat different.  The mutant GABA-Ts were incapable of reacting with DTNB, most likely because of the missing Cys-321 residue. 

To investigate the functional role of GABA-Ts sulfhydryl groups, wild type and mutant enzymes were run on an SDS-PAGE gel in the presence and absence of 2-mercaptoethanol.  In the presence of 2-mercaptoethanol, wild type GABA-T existed as the 50 kDa monomer.  However, in the absence of 2-mercaptoethanol it existed as the 100 kDa dimer.  The mutant enzymes were incapable of forming the dimer and existed as the monomer in both the presence and absence of 2-mercaptoethanol.  The 2-mercaptoethanol is a reducing agent, which reduces the disulfide and generates free sulfhydryl groups.  When 2-mercaptoethanol reacts with the wild type GABA-T, it reduces the disulfide bond that is holding the dimeric structure together and liberates the two monomers.  The mutant GABA-T monomers are both lacking Cys-321; therefore, they have no sulfhydryl groups capable of forming a disulfide bond and will exist only as monomers whether 2-mercaptoethanol is present or not.  Based on these results it is obvious that Cys-321 is involved in a disulfide bond that connects the two monomers of GABA-T and forms the active dimeric enzyme.5
The results of this research have demonstrated that both Lys-357 and Cys-321 are critical for the catalytic activity of human brain GABA-T.  This has given some insight to the structural design of GABA-T.  Based on this information a drug could be designed to either prevent PLP binding or block disulfide formation.  The problem with this is that hundreds of other enzymes are either PLP-dependent or contain disulfide bonds. These enzymes would also be inhibited because the drug would not be specific enough to only inhibit GABA-T.  This makes it clear that the research discussed in this paper is a starting point, and more research is needed before it will be possible to design an effective human GABA-T inhibitor.  The catalytic roles of the Lys and Cys residues are known, but their spatial relationship at the active site is not.  Once the active site structure is determined by X-ray crystallography, it will be easier to propose a mechanism for the conversion of GABA into succinic semialdehyde by GABA-T.  Once a mechanism has been determined, the design of an effective human GABA-T inhibitory drug will be conceivable.  This new drug will be specific to human GABA-T and hopefully, because of this specificity, more effective at preventing epileptic seizures.  If the spatial arrangement of the GABA-T active site or the mechanism by which it acts is unable to be determined, there are other mechanisms available to raise GABA levels.  Both GABA receptors and GABA transporters can be inhibited to keep GABA levels elevated.  However, the receptors and transporters would require the same amount of research to allow for drug development.  In the end, the path taken does not matter; the development of an effective drug to stop seizure activity and improve the quality of epileptic patients' lives is what matters.
 
Works Cited
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2.  Trileptal.  About epilepsy and seizures. < http://www.trileptal.com/info/understanding/about
 _epilepsy.jsp?creative=100002&site=100005&source=01025&campaign=TRI1234>.    [accessed 2005 March 12]

3.  Czuczwar, S.J., Patsalos, P.N. (2001) The new generation of GABA enhancers potential    in the Treatment of epilepsy. CNS Drugs. 15(5), 339-350

4.  Kim, D.W., Yoon C.S., Eum W.S., Lee B.R., An, J.J., Lee, S.H., Lee, S.R., Ahn, J.Y., Kwon,   O.S., Kang, T.C., Won, M.H., Cho, S.W., Lee, K.S., Park, J., Choi, S.Y. (2004) Site-
 directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in 
 cofactor binding at the active site. Mol. Cells. 18, 314-319.

5.  Yoon, C.S., Kim, D.W., Jang, S.H., Lee, B.R., Choi, H.S., Choi, S.H., Kim, S.Y., An, J.J.,   Kwon, O.S., Kang, T.C., Won, M.H., Cho, S.W., Lee, K.S., Park, J., Eum, W.S., Choi,
 S.Y. (2004) Cysteine-321 of human brain GABA transaminase is involved in intersubunit
 cross-linking. Mol. Cells. 18, 214-219

6.  Volkman, Heather. Oklahoma State University Department of Biochemistry and
 Molecular Biology. Purification of GST fusion proteins. . [accessed 2005 March 25]

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