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A Study Of The Enzyme Gaba Transaminase

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

By: Allison Elsey

Class: Chem 4552

Date: April 22,2005

Presented to: Dr. Hobbs

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 е-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



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