Bioc 460 Spring 2008 - Lecture 39 (Miesfeld) Amino Acid Metabolism 2: Amino acid biosynthesis, amino acids are metabolites, metabolic genetic disorders Bioc 460 Spring 2008 - Lecture 39 (Miesfeld) Albinism is caused by mutations in the tyrosinase gene which is required for pigment biosynthesis Roundup Ready soybeans are resistant to glyphosate and are sold by Monsanto It was thought at one time that Count Dracula may have suffered from porphyria

Key Concepts in Amino Acid Metabolism Plants and bacteria synthesize all twenty amino acids, whereas, most other organisms obtain at least some of the required amino acids from their diet. Humans can only synthesize about half of the twenty amino acids. In general, the more complex amino acids are essential amino acids in humans as they require enzymes that have been lost from the human genome over evolutionary time. Phenylalanine is an essential amino acid, but it is also the metabolic precursor to tyrosine through the action of phenylalanine hydroxylase. The herbicide glyphosate (RoundUp) is an inhibitor of the Shikimate Pathway responsible for the synthesis of aromatic amino acids. RoundUp Ready transgenic plants are resistant to the herbicide and are used in agriculture. Numerous genetic diseases caused by defects in amino acid metabolic pathways have been identified. These include phenylketonuria, albinism and porphyrias. Genetic diseases can be due to recessive or dominant mutations.

Overview of Amino Acid Biosynthesis The carbon skeletons of all twenty amino acids are derived from just seven metabolic intermediates, that together, are found in three metabolic pathways. These include: three glycolytic pathway intermediates; 3-phosphoglycerate, phosphoenolypyruvate, and pyruvate, two pentose phosphate pathway intermediates; ribose 5-phosphate and erythrose 4-phosphate two citrate cycle intermediates; -ketoglutarate and oxaloacetate. Note that while plants and bacteria are capable of synthesizing all twenty of the amino acids, most animals are much more restricted in their ability to synthesize amino acids de novo because they lack many of the required enzymes.

Overview of Amino Acid Biosynthesis Based on the principles of nutritional biochemistry, it was determined that humans require ten of the twenty amino acids in their diet in order to thrive. These ten amino acids are called essential amino acids, whereas, the other ten amino acids which humans can synthesize on their own, are called nonessential amino acids.

Overview of Amino Acid Biosynthesis Arginine is listed as an essential amino acid because humans require arginine in their diet to support rapid growth during childhood and pregnancy. However, arginine is actually generated from argininosuccinate in the urea cycle, which means that a small amount of this "essential" amino acid is made available for protein synthesis through this route. Tyrosine is also highlighted because this conditional nonessential amino acid is made in humans from the essential amino acid phenylalanine by the enzyme phenylalanine hydroxylase. Therefore, as long as we have enough phenylalanine in our diets we can generate tyrosine, although in fact, much of the tyrosine in our bodies actually comes directly from dietary tyrosine.

Alanine and aspartate are nonessential amino acids because humans can make them from pyruvate and oxaloacetate, respectively, using transamination reactions. In contrast, essential amino acids, such as tryptophan and methionine, must be obtained from the diet because humans lack the enzymes necessary to synthesize them de novo. In general, the structures of the essential amino acids are more complex than the nonessential amino acids which is reflected in the number of enzymatic reactions required for synthesis.

Since amino acid side groups have different chemical properties, the abundance of individual amino acids in proteins is not uniform. For example, lysine, alanine, valine, isoleucine, glycine and glutamic acid are the most common amino acids in proteins, whereas, cysteine, tyrosine, histidine and methionine are relatively rare. Because of these differences, it is critical that metabolic flux through various amino acid biosynthetic pathways be tightly regulated by feedback inhibition to provide the required proportions of each amino acid in response to cellular needs.

Overview of Amino Acid Biosynthesis The biosynthesis of three nonessential amino acids (alanine, aspartate and asparagine), and six essential amino acids (methionine, threonine, lysine, isoleucine, valine and leucine) in E. coli involves two interconnected pathways utilizing pyruvate and oxaloacetate. The metabolic intermediate -ketobutyrate links the oxaloacetate and pyruvate pathways together because the carbon skeleton of isoleucine is derived from both -ketobutyrate and pyruvate.

Aromatic amino acids are synthesized in plants, fungi, and bacteria by a the shikimate pathway which requires the formation of a C10 compound called chorismate that is the precursor to the three aromatic amino acids tryptophan, tyrosine and phenylalanine. One of the most widely used herbicides is glyphosate, the active ingredient in Roundup®. Glyphosate is a competitive inhibitor of the enzyme EPSP synthase which is required to convert shikimate 3-phosphate to EPSP. Since animals do not have the shikimate pathway enzymes, Roundup is an animal safe herbicide. Do you think glyphosate works faster in the summer or winter? Explain.

Roundup Ready Crops are Glyphosate-Resistant Monsanto developed glyphosate-resistant crop plants so that farmers could spray their transgenic crops with Roundup (also made by Monsanto) and kill weeds that reduce crop yields without harming the crop plants. The first glyphosate-resistant crop plant developed was a strain of soybeans marketed as Roundup Ready® soybeans. In order to protect its investment in the Roundup Ready crop plants, Monsanto sells seeds to the farmers that are sterile so that the transgenic plants cannot be propagated. The farmer must buy seeds from Monsanto. Moreover, with well over 80% of the soybeans planted in the United States being the Roundup Ready variety, the company has a robust market for its many Roundup herbicide products.

Amino Acids as Metabolic Precursors The bulk of amino acids recovered from protein turnover, or obtained from the diet or de novo synthesis, are used to support ongoing protein synthesis in cells. However, because of the nitrogen content of amino acids (the -amino group), they are also used as metabolic precursors for numerous biomolecules, including heme groups (hemoglobin and cytochromes), nucleotide bases (purines and pyrimidines) and a variety of signaling molecules (neurotransmitters, hormones, nitric oxide). For example, the prosthetic group of hemoglobin, myoglobin and cytochromes is heme, a porphyrin ring containing iron.

Amino Acids as Metabolic Precursors Tyrosine is the precursor to several important molecules in metabolic signaling and neurotransmission, including epinephrine and dopamine. Tyrosine is oxidized by the enzyme tyrosine hydroxylase in a reaction requiring the enzyme cofactor tetrahydrobiopterin to form dihydroxyphenylalanine (L-DOPA), a metabolic precursor to dopamine.

Amino Acids as Metabolic Precursors Tyrosine is also the precursor to pigment molecules called melanins that are produced from dopaquinone. The two primary melanins are eumelanins, which are dark pigments having a brown or black color, and pheomelanins that have red or yellow color. The yellow color of pheomelanin pigments comes from the sulfur in cysteine that is combined with dopaquinone.

Amino Acids as Metabolic Precursors Melanocytes are cells that produce melanins, and depending on the ratio of eumelanin and pheomelanin pigments, one can have either dark hair or light hair depending in the distribution of melanin-filled granules along the hair shaft. Natural loss of hair color occurs as a result of aging when melanin production in human melanocytes located near the base of hair follicles shuts down and these defective cells are not replaced as they normally are in younger individuals. Gray hair can be colored by treating it with a mixture of hydrogen peroxide and an ammonia based solution containing artificial pigments.

Inborn Errors of Metabolism: Genetic Disease A genetic defect in the gene encoding phenylalanine hydroxylase is responsible for the metabolic disease phenylketonuria (PKU). Tyrosine is used not only for protein synthesis, but as described above, tyrosine is also the precursor for neurotransmitter dopamine, as well as, skin pigments (melanins) and epinephrine.

Disease symptoms in untreated individuals with PKU include severe mental retardation, stunted growth and dental problems. The clinical symptoms of PKU are caused by the accumulation of phenylalanine in the blood that is 30-50 times higher than normal. This high level of phenylalanine leads to the production of phenylalanine metabolites such as phenylpyruvate, phenylacetate and phenyllactate, all of which are associated with the observed neurological and developmental problems.

NutraSweet contains a phenylalanine derivative Since the symptoms of PKU are caused by excess phenylalanine and its metabolites, and humans require phenylalanine in their diets (it is an essential amino acid), PKU treatment involves careful monitoring of phenylalanine intake to provide just enough for protein synthesis without causing phenylalanine accumulation. Phenylketonuriacs also have to be careful to avoid processed foods and beverages containing the food additive aspartame (aspartyl-phenylalanine methyl ester).

PKU is an autosomal recessive genetic disease The phenylalanine hydroxylase gene is located on chromosome 12 making it an autosomal recessive genetic disease. An autosomal genetic disease is one in which the mutation is located on one of the 22 autosomal chromosomes (all chromosomes except the X or Y chromosome). The probability that two PKU carriers will have a child with the disease is 25% based on simple Mendelian genetics. Since the frequency of PKU carriers in the general population is ~2% (~1 in 50), then the probability that a baby will be born with PKU by random chance is 0.02 x 0.02 x 0.25 = 0.0001, or 1 in 10,000, which is close to the observed frequency of 1 in 15,000. What would the probability be of having a PKU afflicted child if the mother was normal (PP) and the father was a carrier (Pp)?

Type 1 albinism is an autosomal recessive genetic mutation in the tyrosinase gene A deficiency in tyrosinase will result in loss of hair and skin pigments which explains the albino phenotype. Interestingly, individuals with phenylketonuria can have light skin and hair at birth because of low levels of tyrosine. However, phenylketonuriacs are not albinos because they obtain sufficient amounts of tyrosine in their diets to support melanin biosynthesis. Why is PKU treatable, but albinism is not, even though both are the result of genetic mutations in enzymes?

Congenital porphyrias effect heme biosynthesis and can be the result of dominant mutations Numerous metabolic disease affecting heme biosynthesis have been linked directly to enzymes in the heme biosynthetic pathway. These diseases are characterized by the accumulation of heme precursors in the blood and liver and are collectively called porphyrias because they inhibit porphyrin ring synthesis.

Congenital porphyrias effect heme biosynthesis and can be the result of dominant mutations Unlike recessive genetic diseases in which both copies of the gene (maternal and paternal) are defective, dominant genetic diseases cause symptoms when only one of the gene copies is mutated. The inheritance pattern of a dominant genetic disease is such that each child has a 50% chance of being afflicted if one parent carries the mutation and the other parent is normal (100% of the children will get the disease if both parents carry the mutation). How might a mutant protein cause a dominant (gain of function) phenotype?

Congenital porphyrias effect heme biosynthesis and can be the result of dominant mutations It is thought that King George III of England may have suffered from acute intermittent porphyria based on descriptions of his health that were written at the time of the American Revolution. Most notably, he suffered from excruciating stomach pain and episodes of delirium that may have been due to arsenic exposure, one of the environmental factors known to aggravate porphyria symptoms in individuals carrying the defective gene.

Congenital porphyrias effect heme biosynthesis and can be the result of dominant mutations A more rare form of porphyria is congenital erythropoietic porphyria that results from recessive mutations in the gene encoding uroporphyrinogen III cosynthase. This disease is characterized by the accumulation of uroporphyrinogen metabolites that are excreted in the urine giving it a red color. One of the metabolites is uroporphyrinogen I which builds up in the teeth causing them to turn reddish brown and fluoresce under ultraviolet light. These individuals need to avoid excess exposure to sunlight which can result in the formation of painful blisters. The symptoms of congenital erythropoietic porphyria, combined with the common medieval practice of drinking animal blood as a treatment for human ailments, led to the proposal that rare occurrences of porphyria might contribute to the legend of the "vampires."

The Legend of the Vampires Lives on in the Imaginative Minds of Biochemists Everywhere