Czech J. Food Sci. Vol. 24, No. 1: 1–10 Biosynthesis of Food Constituents: Amino Acids: 1. The Glutamic Acid and Aspartic Acid Groups – a Review JAN VELÍŠEK and KAREL CEJPEK Department of Food Chemistry and Analysis, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Prague, Czech Republic Abstract VELÍŠEK J., CEJPEK K. (2006): Biosynthesis of food constituents: Amino acids: 1. The glutamic acid and aspartic acid groups – a review. Czech J. Food Sci., 24: 1–10. This review article gives a survey of principal pathways that lead to the biosynthesis of the proteinogenic amino acids of the glutamic acid group (glutamic acid, glutamine, proline, arginine) and aspartic acid group (aspartic acid, aspar- agine, threonine, methionine, lysine, isoleucine) starting with oxaloacetic acid from the citric acid cycle. There is an extensive use of reaction schemes, sequences, and mechanisms with the enzymes involved and detailed explanations using sound chemical principles and mechanisms. Keywords: biosynthesis; amino acids; glutamic acid; glutamine; proline; arginine; aspartic acid; asparagine; threonine; methionine; lysine; isoleucine Commonly, 20 L-amino acids encoded by DNA aspartic acid, cysteine, glutamic acid, glutamine, represent the building blocks of animal, plant, glycine, proline, serine, tyrosine). Arginine and and microbial proteins, and a limited number histidine are classified as essential, sometimes of amino acids participate in the biosynthesis of as semi-essential amino acids, as their amounts certain shikimate metabolites and particularly in synthesised in the body is not sufficient for normal the formation of alkaloids. The basic amino acids growth of children. Although it is itself non-essen- encountered in proteins are called proteinogenic tial, cysteine (sometimes classified as conditionally amino acids. The biosynthesis of some of these essential amino acid) can partly replace methio- amino acids proceeds by ribosomal processes only nione which is an essential amino acid. Similarly, in microorganisms and plants while the ability tyrosine can partly replace phenylalanine. to synthesise them is lacking in animals includ- ing human beings. These amino acids have to be 1 THE GLUTAMIC ACID GROUP obtained in the diet (or produced by hydrolysis of body proteins) since they are required for normal 1.1 Glutamic acid and glutamine good health; they are referred to as essential amino acids. The essential amino acids are arginine, Free ammonium ions are toxic to living cells and histidine, isoleucine, leucine, lysine, methionine, are rapidly incorporated into organic compounds. phenylalanine, threonine, tryptophan, and valine. One of such transformations is the reaction of The rest of the encoded amino acids are referred to ammonia with 2-oxoglutaric acid from the citric as non-essential amino acids (alanine, asparagine, acid cycle to produce L-glutamic acid. This reac- Partly supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project MSM 6046137305. 1 Vol. 24, No. 1: 1–10 Czech J. Food Sci. Citric acid cycle 2-oxoglutaric acid L-glutamic acid L-ornithine L-arginine L-glutamine L-proline L-citrulline Figure 1 tion is known as reductive amination. Glutamic catalysed by glutamine synthetase (EC 6.3.1.2) and acid is, accordingly, the first amino acid generated. proceeds through the intermediate L-glutam-5-yl Glutamic acid itself can bind a further ammonium phosphate-ATP which splits off ADP and phos- ion to form L-glutamine, the second amino acid. phoric acid (Figure 3). Glutamic acid then serves as the precursor of Very important for many amino acid syntheses L-proline, L-ornithine, L-citrulline, and L-argi- is the group of enzymes called transaminases. nine. The biosynthetic pathway of the glutamic They are able to transfer an amino group (mostly acid amino acid family is schematically shown in that of glutamine) to a 2-oxo acid according to the Figure 1 (SENGBUSCH). following equation: The formation of glutamic acid (as well as the reverse rection) is catalysed by glutamate dehy- (amino acid)1 + (2-oxo acid)2 = (2-oxo acid)1+ drogenase. Nicotinamide adenine dinucleotide + (amino acid)2 (NADH)-dependent glutamate dehydrogenase (EC Thus, the glutamine amino group is transferred 1.4.1.2) or the enzyme using nicotinamide adenine to 2-oxoglutaric acid (Figure 4) producing glutamic dinucleotide phosphate (NADPH) as the coenzyme acid. The reaction is catalysed by glutamate syn- (EC 1.4.1.4) is employed as hydrogen donor by thases having either NADPH (EC 1.4.1.13) or microorganisms (e.g. Saccharomyces cerevisiae). NADH (EC 1.4.1.14) as cofactor. 1 In plants is the reaction mainly dependent on the The transamination reaction catalysed by trans- coenzyme NAD(P)H (EC 1.4.1.3) (Figure 2). aminases is dependent on the coenzyme pyridoxal L-Glutamine arises as a product of amidation of 5'-phosphate (PLP). The reaction starts with the glutamic acid that binds a further ammonium ion addition of the unprotonised amino group of an (SENGBUSCH). This amidation of glutamic acid is amino acid to the electron deficient carbon of NAD(P)H + H NADP HOOC COOH HOOC COOH + H O + NH3 2 O EC 1.4.1.3 NH2 2-oxoglutaric acid L-glutamic acid Figure 2 ADP ATP NH3 P O O HOOC COOH COOH PO COOH H2N NH2 EC 6.3.1.2 NH2 EC 6.3.1.2 NH2 L-glutamic acid L-glutam-5-yl phosphate L-glutamine Figure 3 1Plants take up nitrogen as nitrates and in smaller amounts also as ammonium ions. A few species, mainly leguminose plants, live in symbiosis with nitrogen-fixing bacteria that are able to reduce atmospheric nitrogen. Microorganisms mainly require ammonium ions as the nitrogen source, some bacteria and moulds are able to use nitrates similarly to plants. Plants reduce nitrates in two steps. In the first step, nitrate is reduced to nitrite by nitrate reductase (NADH) (EC 1.7.1.1). In the second step, nitrite is reduced to ammonia by ferredoxin-nitrite reductase (EC 1.7.7.1). 2 Czech J. Food Sci. Vol. 24, No. 1: 1–10 NADPH + H O NADP COOH HOOC COOH HOOC COOH HOOC COOH H N 2 + + NH2 O NH NH EC 1.4.1.13 2 2 L-glutamine 2-oxoglutaric acid L-glutamic acid L-glutamic acid Figure 4 the polarised carbonyl group of PLP under the 5-semialdehyde formed yields spontaneously a formation of a monotopic carbinolamine. The cyclic Schiff base (S)-1-pyrroline-5-carboxylic carbinolamine dehydration then leads to an aldi- acid, i.e. (S)-3,4-dihydro-2H-pyrrole-2-carboxylic mine (Schiff base). The α-hydrogen atom of the acid. Its reduction to proline is achieved by pyr- original amino acid in the aldimine is now much roline-5-carboxylate reductase (EC 1.5.1.2) and more acidic and can be readily eliminated. Isom- the hydrogen needed is supplied by NAD(P)H. erisation of the resulting intermediate leads to a An alternative pathway of proline biosynthesis is ketimine (an imine of PLP with an 2-oxo acid). a cyclisation of L-ornithine catalysed by ornithine Hydrolysis of this ketimine generates pyridoxamine cyclodeamidase (EC 4.3.1.12) (Figure 6). 5'-phosphate and the 2-oxo acid (Figure 5). The amino group of pyridoxamine 5'-phosphate is then 1.3 Arginine transferred to another 2-oxo acid molecule and PLP is regenerated (DEWICK 2002). The biosynthesis of the essential amino acid L-arginine in microorganisms and plants starts 1.2 Proline from L-glutamic acid (IUBMB 2001). It proceeds via L-ornithine in several intermediate steps and L-Proline is generated from L-glutamic acid by needs the reduction of the γ-carboxyl group to a ring formation (IUBMB 2001). The reaction is carbonyl group under the participation of ATP facilitated by a preliminary phosphorylation of similarly to proline biosynthesis. To prevent the glutamic acid by γ-glutamyl kinase (EC 2.7.2.11) spontaneous cyclisation of the semialdehyde, the that leads to an unstable intermediate L-glutam-5-yl first step is acetylation of glutamic acid with acetyl- phosphate under consumption of one molecule CoA catalysed by amino-acid N-acetyltransferase of ATP. The reduction of the phosphorylated (EC 2.3.1.1). The carboxyl group of the intermediate γ-carboxyl to a carbonyl group is catalysed by N-acetyl-L-glutam-5-yl phosphate thus formed is the NAD(P)H dependent glutamate semialde- then reduced to a carbonyl group by NADPH-de- hyde dehydrogenase (EC 1.2.1.41). Glutamic acid pendent N-acetyl-γ-glutamyl-phosphate reductase R COOH R COOH H H O HO NH N OH OH OH R COOH PO PO PO + H2O H + NH2 N CH3 N CH3 N CH3 H 2-amino acid pyridoxal 5´-phosphate vicinal carbinolamine aldimine R COOH R COOH NH2 N H N OH OH OH R COOH PO PO PO + O N CH N CH 3 3 N CH3 H 2-oxo acid pyridoxamine 5´-phosphate ketimine Figure 5 3 Vol. 24, No. 1: 1–10 Czech J. Food Sci. ATP ADP NAD(P)H NAD(P) + P O H HOOC COOH PO COOH O COOH NH 2 EC 2.7.2.11 NH2 EC 1.2.1.41 NH2 L-glutamic acid L-glutam-5-yl phosphate L-glutamic acid 5-semialdehyde NH3 2 NAD(P) 2 NAD(P)H H2O COOH H2N H H N COOH NH2 H N COOH EC 1.5.1.2 EC 4.3.1.12 H L-ornithine L-proline (S)-1-pyrroline-5-carboxylic acid Figure 6 (EC 1.2.1.38) and the next intermediate N-acetyl- L-argininosuccinic acid. Argininosuccinate lyase L-glutamic acid 5-semialdehyde is transformed to (EC 4.3.2.1) then splits this intermediate to arginine N-acetyl-L-ornithine in a transamination reaction and fumaric acid (Figure 8).