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A single mutation in an enzyme can lead to a fatal disease

04/07/2015 Dr. Arnthor Ævarsson Ph.D., The Enzyme Blog

Mutations in enzymes can lead to serious or fatal disorders in humans and are the consequence of inherited abnormalities in the DNA of the affected individual. The mutation may be just as a single abnormal amino acid residue at a specific position in an enzyme encoded by a mutated gene. An example of such an inherited genetic disorder is Maple Syrup Urine Disease (MSUD), also called branched-chain ketoaciduria.

The classic form of the disease is manifested within the first weeks of life and if untreated leads to seizures, coma and finally death. Milder forms of MSUD become apparent later in life and may involve developmental delay and mental retardation. The name of the disease and reference to maple syrup comes from the distinctive sweet odour of the patient’s urine due to the fact that the or she is not able to digest certain amino acid found in normal food which leads to toxic accumulation of the corresponding acids in the body and the ultimate consequences of the disease. The disorder affects only about 1 in 185.000 infants worldwide on average but in certain populations the disease is much more prevalent such as in the population of the Old Order Mennonites with an estimated incidence of about 1 in 380 newborns.

The branched-chain amino-acids Valine, Leucine and Isoleucine that are present in many kinds of food are normally broken down in the body down by a sophisticated and very large multi-enzyme complex called Branched-chain alpha-keto acid (2-oxo acid) dehydrogenase complex. The complex is formed my multiple copies of three types of enzymes referred to as E1, E2 and E3. A Dihydrolipoyl transacetylase (E2) forms the core of the complex with numerous copies of Branched-chain alpha-keto acid dehydrogenase (E1) and Dihydrolipoamide dehydrogenase (E3) attached to the E2 core. The different specific activities of these enzymes and the intricate interplay between the enzyme in the complex ensures that these amino acid in the human diet are properly metabolised in a series of enzymatic steps.

Animation of Human Branched-chain Alpha-keto acid Dehydrogenase

Human Branched-chain Alpha-keto acid Dehydrogenase (E1)

The human genes encoding the proteins that constitute the E1 component of the complex have been found to be the location of many identified mutations that cause MSUD. In a collaboration between research groups at the University of Texas, Southwestern Medical Center and University of Washington School of Medicine, the three-dimensional structure of the Human Branched-chain alpha-keto acid dehydrogenase (E1) was determined (1) in order to gain insight into the molecular basis of the disease. The enzyme, shown here in the animation, is formed by two different types of protein chains, alpha (green and cyan colour) and beta (yellow and magenta colour), each found in two copies and forming an enzyme with 4 subunits (tetramer, α2β2). The two active sites are buried within the enzyme at the border between subunit as indicated by the bound cofactor Thiamin diphosphate (i.e. Vitamin B1) shown here in a multi-colored space-filling model. The enzyme also binds a few Potassium ions (K+) which seem important for the integrity of the structure (purple spheres).

The structure provided the opportunity to look specifically at the location of each known mutation to model the abnormal amino acid residues caused by the respective mutation and predict the consequences for the structure and function of the enzyme. Some of the known MSUD mutations seem to interfere with the cofactor or potassium ion binding while others seem to disrupt the hydrophobic core and may cause improper folding of the alpha subunits. However, it is striking to see that many of the mutations cluster at interfaces between subunits and apparently interfere with normal interaction between subunits. This includes the classic “Mennonite mutation” with a Tyrosine residue substituted by an Asparagine residue at position 393 (Y393N) in a unique extension of the alpha subunit contacting a beta subunit. It seems that this class of mutations may prevent or cause an improper and unstable assembly of the subunits into the tetrameric enzyme. It is clear from the structure that a fully functional enzyme can only be formed by the correct and stable assembly of the subunits since the active site is formed by residues from both the alpha and the beta subunits.

For some of the milder mutations that possibly may not prevent but lead to unstable assembly of the subunits, the structure may lend itself to the discovery of compounds that could be therapeutically useful for the less severe forms of maple syrup urine disease. Small compounds that could bind to the enzyme and increase its stability such as by binding across subunit interfaces could be a starting point for development of such therapeutic agents.

1. Ævarsson et al. 2000. Crystal structure of the human branched-chain α-ketoacid dehydrogenase and the molecular basis of maple syrup urine disease. Structure 8:277-291

3. Protein Data Bank Entry 1DTW