Structure and Mechanism
Beta-Hexosaminidase A is an enzyme that is expressed in all cells of the body, including cells in the brain and nervous system, spleen and GI tract. Beta-Hexosaminidase A uses a series of nucleophilic attacks to remove the terminal Beta linked GalNAc from ganglioside GM2 to create gangliosides GM3 in the lysosome, this is one step in the process used to degrade gangliosides. When this mechanism fails it can create a group of diseases called Tay-Sachs. The most severe form of which being infantile Tay-sachs. This paper will discuss the structure of Beta-Hexosaminidase A, how it works to degrade gangliosides, the symptoms and trends of Tay-sachs disease, and what treatments are currently being looked into treat it.
Structure and Mechanism
Beta-Hexosaminidase A is a protein that contains two distinct subunits the alpha and the beta subunit. Each subunit also contains 2 domains. One major difference between the two units is that alpha subunit contains a flexible alpha280GSEP283 loop, this loop is important because it allows for the interaction between helper protein that presents GM2 and Beta-Hexosaminidase A, and is cleaved post-translationaly in the beta subunit.. Each subunit contains two active sites allowing the enzyme to bond two substrates at once. The active sites are located at the opening of the TIM barrels where the alpha and beta subunit meet. Beta-Hexosaminidase A is a hetrodimer, meaning that the two units interact with each other to provide stability. (1)
The mechanism for the reduction of GM2 to GM3 starts with the binding of a helper protein called GM2 ganglioside activator pseudogene (GM2AP), which can be seen in the Jmol viewer. GM2AP is a transport protein with a deep hydrophobic pocket that binds GM2. This protein transfer the GM2 that it has bound into the active site of Beta-Hexosaminidase A (2). Inside the active site of the Beta-Hexosaminidase A the Glutamate residue creates on the Beta subunit creates a hydrogen bond with the nitrogen atom on GalNAc. This stabilizes the GM2 while the reaction takes place and holds the structure in place. At the same time that the Beta-subunit bonds to the nitrogen, the aspartic acid has a nucleophilic attach on the carbon connected to the GM3 group. The attack follows a reaction in which the HOR group is cleaved at the same time the carbon in the prynose ring forms bonds to the remaining oxygen forming a ring structure, this is also known as a SN2 reaction and the resulting product is a GM3 that is then released back into the lysosome. Next a water molecule moves in and the negatively charged glutamatic acid takes hydrogen from the water creating a negative charge on the oxygen. At the same time the C2 carbon attacks the oxygen on the water. This process creates a stable leaving group that can now be released from the enzyme and reforms the active site (1).See figure 1 for a diagram of this mechanism, or the Jmol active site feature for a better understanding of the placement of the active site.
Gangliosides are part of a group known as the sphingolipids they are type of phospholipid containing a sphingosine backbone rather then, the more common glycerol backbone. They also contain at least one sialic acid group linked to the terminal hydroxide group. Figure 2 shows the structure of a GM2 ganglioside.
They have many important functions in the body, it has been well known for some time that gangliosides are present in large quantities in the brain before birth, and that they play a role in the development and differentiation of the central nervous system in vertebrates. It is also known that this compound is present in the cell membrane of all Eukaryotic cells, with a higher concentration in cells of the central nervous system (3). However, more information has recently become available proving that the compound has a much bigger role in the maintenance of tissue and repair after injury of brain tissue. An experiment done at the Nagoya University found that mice who had the gene removed for the production of gangliosides suffered neurodegeneration starting from birth and progressing with age. They also were noted to have a deregulation of the compliment system with inflammation in their brains; this kind of inflammation is also commonly seen in Alzheimer's patients (4).
Ganglosides do serve a function in the cell, but their storage can also cause problems, one of those problems is called Tay-Sachs. Although there are three forms of Tay-Sachs this paper will only take about the form known as infantile Tay-Sachs. Infantile Tay-Sachs is the most extreme form of the disease. Babies with Tay-sachs start out life just as any other baby, developing normally for the first four to six months of their life. The first warning sign many parents notice is the presence of bright cherry red spots in the babies' eye. It is at this time that infants are commonly brought in for testing. Then neurological symptoms begin to appear, the child loses skills they had already developed, such as holding their head up, rolling over, and recognizing their parents (5). This neurological backslide is caused by the failure of Beta-Hexosaminidase A. Without Beta-Hexosaminidase A degrading GM2 to GM3 the substance accumulates in the lysosomes. Tay-Sachs usually expresses itself in the nerve cells, most importantly in the central nervous system. As these nerve cells accumulate the GM2 they begin to bulge, and eventually split open and die. This process of cell death leads to the neurological symptoms in these patients (1). As the disease progresses patients experience blindness, deafness, and seizures. Patients typically die before age four (5).
Infantile Tay-sachs is a heredity disease which can be caused by a variety of mutations, all of which are caused by changes in this enzyme the cause it to not cease functioning. The most common mutation is a four base insertion TATC found in exon 11 of the HEXA gene. This mutation creates and early stop codon and is found in approximately 80% of all infantile Tay-sachs cases (6). Figure 3 shows the location of all known mutations to Beta-Hexosaminidase A
Tay-sachs is more common in some communities in others. For the average person the rate of carriers is about 1 in every 300 people, however for Ashkenazi Jews about 1 in 29 people are carriers and 1 out of every 3,000 babies are affected (7). There are several theories for why this is the case. One reason is that these groups are more isolated breeding; most Ashkenazi Jews try to find and marry another person from this group increasing the odds of spreading this trait through the group. Another reason that has been theorized is that when a parent loses a child or many children to this disease they compensate by having many more babies, the side effect this type of breeding is that more carriers are produced increasing their percentage in their isolated populations. However, although carrier status is higher among these groups' actual cases of the disease are on the decline. This is due mostly to concentrated efforts by members of the community to raise awareness and encourage testing for carrier status before people get married (8)(4).
There is no cure for Tay-sachs disease, much of the care these patients receive is designed around making them more comfortable for what time they have. However, new techniques are being investigated currently to work at either treating or curing the disease. Some of the treatments currently being researched are Substrate Reduction Therapy, Enzyme Replacement Therapy, Chaperone chemicals, and Gene Therapy. For the purposes of this paper however we will only be taking about Enzyme Replacement Therapy, because this is the one that science is the closet to achieving (9).
The basic concept behind enzyme replacement therapy is actually relatively simple. It involves either injecting the enzyme so that the cells can incorporate the whole enzyme, or injecting the component parts so that the cell can incorporate them and then form the new enzyme (10). There is one lysosomial storage disorder being treated with this method, Gauchers disease type I. Gacuhers disorder is similar to Tay-Sachs in many ways. It is caused by the lack of the enzyme Beta-glucocerebrosidase, without this enzyme patients suffer from a buildup of a substance known as glucocerebroside in the lysosomes of the cells in the liver, spleen, and bone marrow. These cells cease to function themselves and also displace other healthy functioning tissue (11). Cerezyme, is a drug currently being used as an enzyme replacement to treat Gaucher's disease it works by creating a surplus of imiglucerase a substance that is analogous for glucocerebrosidase. Once in the cell this enzyme takes over the function of the missing enzyme and in many cases eliminates the symptoms caused by the disorder (12). There are however a few important difference between Tay-Sachs and Gauchers that makes Gauchers much more treatable.
One reason that attempts at treating Gauchers have been more successful then attempts at treating Tay-Sachs is because of the placement of the cells involved. In Gauchers disease the cells involved are in places that see a lot of blood flow and have little protection from compounds in the blood stream. This is not true for Tay-Sachs, Tay-Sachs involves the cells of the central nervous system meaning that for an enzyme to be effective it would have to cross the blood brain barrier and cross in high enough concentrations to be effective. This limitation is also the reason why only type I Gauchers can be treated, it involves no neurological symptoms. The second problem faced when using enzyme replacement therapy to treat Tay-Sach's disease is the size of the enzyme involved. Beta-glucocerebrosidase is only 492 residues long, while Beta-Hexosaminidase A is more than twice as big at 1014 residues. This means that if it ever becomes possible to get the enzyme to the cells that need it, it will still be difficult to get the compound past the cell membrane and into the cells themselves. These two major complications mean that are still many steps that need to be taken before an effective treatment for Tay-Sachs is in use (10).
In conclusion Beta-Hexosaminidase A is an enzyme that uses a series of SN2 reactions and polar bonds in its active site to hydrolyze GM2 to GM3. It is a necessary enzyme for human life and its absence leads to the disease known as Tay-Sachs. Tay-Sachs affects certain minorities more than others, but amongst groups where it is a special concern community effort has lead to an actual drop in the disease state, although the unusually high amount of carriers remains. Although treatments for a disease like this are possibly, due to specific limitations in the location of the problem and size of the enzyme it is still not possible to treat Tay-Sachs, although new research into this area brings hope for the patients and families who suffer due to mutations in this enzyme..
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