DOCSLIB.ORG

Amino Acids: 1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Amino Acids: 1 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).
Load more

Recommended publications
  • Namely, Aspartokinase and Aspartate 3-Semialdehyde Dehydrogenase) Are Not Subject to Feedback Inhibition Control by the End Product Methionine
    REGULATION OF HOMOSERINE BIOSYNTHESIS BY L-CYSTEINE, A TERMINA L METABOLITE OF A LINKED PA THWA Y* By PRASANTA DATTA DEPARTMENT OF BIOLOGICAL CHEMISTRY, UNIVERSITY OF MICHIGAN, ANN ARBOR Communicated by Martin D. Kamen, June 23, 1967 In bacteria, the amino acid homoserine is a key branch-point intermediate in the synthesis of several amino acids of the aspartic pathway.' On the one hand, homoserine is converted to threonine (and thus to isoleucine), and through a separate sequence of reactions is transformed to methionine.1 In the latter se- quence, a succinylated product of homoserine is condensed with cysteine to pro- duce cystathionine,2-5 a precursor of methionine. Recent studies (see refs. 6 and 7 for up-to-date reviews on the subject) with several microorganisms have revealed that the synthesis of homoserine is regulated by various combinations of repression and/or feedback inhibition controls of early biosynthetic enzymes of the aspartic pathway by several end-product metabolites. One of these enzymes, homoserine dehydrogenase, catalyzes the pyridine nucleotide-linked reduction of aspartate j3-semialdehyde to homoserine. In the various bacteria that have been examined, this dehydrogenase as well as the two earlier enzymes of the path- way (namely, aspartokinase and aspartate 3-semialdehyde dehydrogenase) are not subject to feedback inhibition control by the end product methionine. This report describes the results of experiments on the control of activity of several bacterial homoserine dehydrogenases by L-cysteine, a terminal metabolite of a linked or connecting pathway, required for the synthesis of cystathionine.2-5 The findings suggest that the size of the homoserine pool in bacterial cells must depend, at least in part, on complex interdependent regulatory interactions of both cysteine and threonine on homoserine dehydrogenase.
    [Show full text]
  • Internal Homologies in the Two Aspartokinase-Homoserine
    Proc. Nati. Acad. Sci. USA Vol. 81, pp. 3019-3023, May 1984 Biochemistry Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12 (gene duplication/evolution/bifunctional proteins) PASCUAL FERRARA, NATHALIE DUCHANGE, MARIO M. ZAKIN, AND GEORGES N. COHEN The Unite de Biochimie Cellulaire, Ddpartement de Biochimie et Gdndtique Moldculaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France Communicated by Norman H. Horowitz, January 27, 1984 ABSTRACT In Escherichia coli, AK I-HDH I and AK II- METHODS HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the com- Protein sequence comparisons were done by computer. mon pathway leading to threonine and methionine. An exten- Each sequence was compared with itself by a simple frame- sive amino acid sequence comparison of both molecules reveals shift method using different segment lengths. After small ho- two main features on each of them: (i) two segments, each of mologous fragments were found, comparison was extended about 130 amino acids, covering the first one-third of the poly- in both directions, allowing gaps to maximize homologies. peptide chain, are similar to each other and (ii) two segments, Finally, the fragments were aligned with a computer pro- each of about 250 amino acids and covering the COOH-termi- gram, provided by T. F. Smith and W. M. Fitch, using the nal 500 amino acids also present a significant homology. These matrix algorithm of Sellers (8), modified by Smith et al. (9). findings suggest that these two regions may have evolved inde- A deletion weight of 0.7 for each gap plus 1.5 times the pendently of each other by a process of gene duplication and number of residues in each gap was initially used.
    [Show full text]
  • The Biosynthesis of Cystathionine in Patients with Homocystinuria
    Pediat. Res. 2: 149-160 (1968) Cystathionase homoserine cystathionine pyridoxine cystine serine homocystinuria The Biosynthesis of Cystathionine in Patients with Homocystinuria P.W.K.WONG[30], V.SCHWARZ and G.M.KOMROWER Department of Medical Biochemistry, University of Manchester, and the Mental Retardation Unit, Royal Manchester Children's Hospital, Manchester, England Extract The synthesis in rat and normal human liver homogenates of cystathionine from homoserine +cysteine was studied. When cystathionine was used as substrate, cyst(e)ine was formed and incubation of liver homogenate with homoserine-(-cysteine resulted in the formation of cystathionine. Incubation of homogenate prepared from homocystinuric hVer with L-homoserine+DL-cysteine- 3-14G or DL-cysteine-35S resulted in the formation of labelled cystathionine. The identity of cystathio- nine was established in three chromatographic separations. Oral loading with homoserine and cysteine or cystine in two patients with homocystinuria resulted in urinary excretion of cystathionine in amounts similar to that reported in normal human subjects without amino acid loading. Speculation The observation that human and monkey brains contain much larger quantities of cystathionine than those of other species [26] suggests a special relation of this amino acid to the normal development and function of the primate brain. Cystathionine is either absent from or markedly deficient in the brain of untreated homocystinuric patients [2, 9]. Since we have shown that synthesis of cystathionine from homoserine and cysteine does take place in the patient's liver in vitro, supplementation of the diet with homoserine and cysteine may prove effective in raising intracellular cystathionine concentration to- ward the normal, and may possibly improve the prognosis in this disease.
    [Show full text]
  • Purification and Characterization of the Higher Plant Enzyme L-Canaline Reductase (L-Canavanine Catabolism/Plant Nitrogen Metabolism/Leguminosae) GERALD A
    Proc. Natd. Acad. Sci. USA Vol. 89, pp. 1780-1784, March 1992 Biochemistry Purification and characterization of the higher plant enzyme L-canaline reductase (L-canavanine catabolism/plant nitrogen metabolism/Leguminosae) GERALD A. ROSENTHAL T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, KY 40506 Communicated by John S. Boyer, December 3, 1991 (receivedfor review April 20, 1991) ABSTRACT A newly discovered enzyme, L-canaline re- oglutaric acid to generate stoichiometrically a canaline-2- ductase (NADPH:L-canaline oxidoreductase, EC 1.6.6.-), has oxoglutaric acid oxime (5). Canaline also reacts readily with been isolated and purified from 10-day-old leaves of the jack the pyridoxal phosphate moiety of vitamin B6-containing bean Canavalia ensiformis (Leguminosae). This higher plant is enzymes to form a stable, covalently linked complex (6, 7). representative of a large number of legumes that synthesize In vitro analysis of canaline interaction with homogeneous L-canavanine, an important nitrogen-storing nonprotein amino ornithine aminotransferase (ornithine-oxo-acid aminotrans- acid. Canavanine-storing legumes contain arginase, which ferase; L-ornithine:2-oxo-acid aminotransferase, EC hydrolyzes L-canavanine to form the toxic metabolite L-cana- 2.6.1.13) reveals its marked ability to form an oxime complex line. Canaline reductase, having a mass of =167 kDa and with and thereby inhibit this pyridoxal phosphate-dependent composed of 82-kDa dimers, catalyzes a NADPH-dependent enzyme (8). As little as 10 nM canaline causes a significant reductive cleavage of L-canaline to L-homoserine and ammo- reduction in ornithine aminotransferase activity (9). nia. This is the only enzyme known to use reduced NADP to Canavanine-storing legumes can accumulate high levels of cleave an O-N bond.
    [Show full text]
  • Fortifying Horticultural Crops with Essential Amino Acids: a Review
    Review Fortifying Horticultural Crops with Essential Amino Acids: A Review Guoping Wang 1, Mengyun Xu 1, Wenyi Wang 1,2,* and Gad Galili 2,* 1 College of Horticulture, South China Agricultural University, Guangzhou 510642, China; [email protected] (G.W.); [email protected] (M.X.) 2 Department of Plant Science, Weizmann Institute of Science, Rehovot 76100, Israel * Corresponding author: [email protected] (W.W.); [email protected] (G.G.); Tel.: +972-8-934-3511 (G.G.); Fax: +972-8-934-4181 (G.G.) Received: 17 May 2017; Accepted: 14 June 2017; Published: 19 June 2017 Abstract: To feed the world′s growing population, increasing the yield of crops is not the only important factor, improving crop quality is also important, and it presents a significant challenge. Among the important crops, horticultural crops (particularly fruits and vegetables) provide numerous health compounds, such as vitamins, antioxidants, and amino acids. Essential amino acids are those that cannot be produced by the organism and, therefore, must be obtained from diet, particularly from meat, eggs, and milk, as well as a variety of plants. Extensive efforts have been devoted to increasing the levels of essential amino acids in plants. Yet, these efforts have been met with very little success due to the limited genetic resources for plant breeding and because high essential amino acid content is generally accompanied by limited plant growth. With a deep understanding of the biosynthetic pathways of essential amino acids and their interactions with the regulatory networks in plants, it should be possible to use genetic engineering to improve the essential amino acid content of horticultural plants, rendering these plants more nutritionally favorable crops.
    [Show full text]
  • A Non-Derivatized Method for Simultaneous Quantitation of Proteinogenic, Urea-Cycle, and Acetylated Amino Acids by Liquid Chroma
    Environmental Chemistry Letters (2020) 18:229–235 https://doi.org/10.1007/s10311-019-00927-4 ORIGINAL PAPER A non‑derivatized method for simultaneous quantitation of proteinogenic, urea‑cycle, and acetylated amino acids by liquid chromatography–high‑resolution mass spectrometry Annaleise R. Klein1,2 · Krista A. Barzen‑Hanson1,3 · Ludmilla Aristilde1,2 Received: 27 July 2019 / Accepted: 8 August 2019 / Published online: 24 August 2019 © Springer Nature Switzerland AG 2019 Abstract The quantifcation of amino acids as freely dissolved compounds or from hydrolyzed peptides and proteins is a common endeavor in biomedical and environmental sciences. In order to avoid the drawbacks of derivatization and application chal- lenges of tandem mass spectrometry, we present here a robust 13-min liquid chromatography coupled with a full-scan mass spectrometry method to achieve rapid detection and quantifcation of 30 amino acids without derivatization. We combined hydrophilic interaction liquid chromatography with heated electrospray ionization and high-resolution mass spectrometry operated with polarity switching to analyze the 20 proteinogenic amino acids, ornithine, citrulline, homoserine, cystine, and six acetylated amino acids. We obtained high mass accuracy and good precision of the targeted amino acids. Limits of detection ranged from 0.017 to 1.3 µM. Noteworthy for environmental samples, we found comparable ionization efciency and quantitative detection for the majority of the analytes prepared with pure water versus a high-salt solution. We applied the method to profle carbon source-dependent secretions of amino acids by Pseudomonas protegens Pf-5, a well-known plant-benefcial bacterium. Keywords Polarity switching · Non-targeted analysis · Acetylated amino acids · Urea-cycle amino acids · High-resolution mass spectrometry Introduction 2012), physiological responses to toxicity (Dubey et al.
    [Show full text]
  • Method for the Affinity Purification of Covalently Linked Peptides
    Anal. Chem. 2009, 81, 9885–9895 Method for the Affinity Purification of Covalently Linked Peptides Following Cyanogen Bromide Cleavage of Proteins Tujin Shi,† Rasanjala Weerasekera,†,‡ Chen Yan,‡ William Reginold,‡ Haydn Ball,§ Thomas Kislinger,| and Gerold Schmitt-Ulms*,†,‡ Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas, and Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada The low resolution structure of a protein can sometimes posttranslational modifications,6 and the mapping of protein-nucleic be inferred from information about existing disulfide acid interactions are increasingly well-developed,7-13 gaining bridges or experimentally introduced chemical crosslinks. insights into the folds of proteins and contact sites between Frequently, this task involves enzymatic digestion of a proteins remains a challenging undertaking. Despite major protein followed by mass spectrometry-based identifica- advances in speed and application range, high-resolution structure tion of covalently linked peptides. To facilitate this task, methods based on X-ray crystallography or nuclear magnetic we developed a method for the enrichment of covalently resonance (NMR) analyses continue to be time-consuming and linked peptides following the chemical cleavage of a are limited by the need to obtain analytes at high levels of purity protein. The method capitalizes on the availability of and quantity. The quest for a robust methodology that fills this homoserine lactone moieties at the C-termini of cyanogen need has arguably become one of the most pressing problems in bromide cleavage products which support selective con- protein science.
    [Show full text]
  • A New Family of Extraterrestrial Amino Acids in the Murchison Meteorite
    www.nature.com/scientificreports OPEN A new family of extraterrestrial amino acids in the Murchison meteorite Received: 16 November 2016 Toshiki Koga1 & Hiroshi Naraoka1,2 Accepted: 8 March 2017 The occurrence of extraterrestrial organic compounds is a key for understanding prebiotic organic Published: xx xx xxxx synthesis in the universe. In particular, amino acids have been studied in carbonaceous meteorites for almost 50 years. Here we report ten new amino acids identified in the Murchison meteorite, including a new family of nine hydroxy amino acids. The discovery of mostly C3 and C4 structural isomers of hydroxy amino acids provides insight into the mechanisms of extraterrestrial synthesis of organic compounds. A complementary experiment suggests that these compounds could be produced from aldehydes and ammonia on the meteorite parent body. This study indicates that the meteoritic amino acids could be synthesized by mechanisms in addition to the Strecker reaction, which has been proposed to be the main synthetic pathway to produce amino acids. The extraterrestrial synthesis of amino acids is an intriguing discussion concerning the chemical evolution for the origins of life in the universe, because amino acids are fundamental building blocks of terrestrial life. The extrater- restrial amino acid distribution has been extensively examined using carbonaceous chondrites, which are the most chemically primitive meteorites containing volatile components such as water and organic matter, particularly the Murchison meteorite since it’s fall in 1969. The Murchison meteorite is classified as a CM2 (Mighei-type) chon- drite, moderately altered by aqueous activity on the parent body (e.g. ref. 1). Currently, a total of 86 amino acids have been identified in the Murchison meteorite as α, β, γ and δ amino structures with a carbon number between 2–4 C2 and C9 including dicarboxyl and diamino functional groups .
    [Show full text]
  • Partial Purification and Some Properties of Homoserine O
    JOURNAL OF BACTERIOLOGY, Aug. 1987, p. 3458-3463 Vol. 169, No. 8 0021-9193/87/083458-06$02.00/0 Copyright X) 1987, American Society for Microbiology Partial Purification and Some Properties of Homoserine O-Acetyltransferase of a Methionine Auxotroph of Saccharomyces cerevisiae SHUZO YAMAGATA Department ofBiology, Faculty of General Education, Gifu University, Gifu 501-11, Japan Received 15 December 1986/Accepted 27 April 1987 A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four muitant strains exhibited enzyme activity in cell extracts, Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strkin through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, Phenyl- Sepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal p1I at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was esimated to be approximately 104,000 by Sephadex G-150 column chromatography anid 101,000 by sucrose density gradient centrifugation.
    [Show full text]
  • Inhibition of Homoserine Dehydrogenase by Formation of A
    www.nature.com/scientificreports OPEN Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex Received: 30 October 2017 Kohei Ogata1, Yui Yajima2, Sanenori Nakamura3, Ryosuke Kaneko1, Masaru Goto1, Accepted: 27 March 2018 Toshihisa Ohshima4 & Kazuaki Yoshimune2,3 Published: xx xx xxxx Homoserine dehydrogenase (EC 1.1.1.3, HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate. Here, HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) was found to be inhibited by cysteine, which acted as a competitive inhibitor of homoserine with a Ki of 11 μM and uncompetitive an inhibitor of NAD and NADP with Ki’s of 0.55 and 1.2 mM, respectively. Initial velocity and product (NADH) inhibition analyses of homoserine oxidation indicated that StHSD frst binds NAD and then homoserine through a sequentially ordered mechanism. This suggests that feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Structural analysis of StHSD complexed with cysteine and NAD revealed that cysteine situates within the homoserine binding site. The distance between the sulfur atom of cysteine and the C4 atom of the nicotinamide ring was approximately 1.9 Å, close enough to form a covalent bond. The UV absorption- diference spectrum of StHSD with and without cysteine in the presence of NAD, exhibited a peak at 325 nm, which also suggests formation of a covalent bond between cysteine and the nicotinamide ring. Among the various amino acid metabolic pathways found in plants and most bacterial strains, the aspartate path- way is pivotal because it is responsible for the production of multiple amino acids, including lysine, threonine, methionine, and isoleucine1.
    [Show full text]
  • Novel Regulatory Mechanism of Serine Biosynthesis Associated with 3-Phosphoglycerate Dehydrogenase in Arabidopsis Thaliana
    www.nature.com/scientificreports OPEN Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate Received: 21 December 2016 Accepted: 17 May 2017 dehydrogenase in Arabidopsis Published: xx xx xxxx thaliana Eiji Okamura & Masami Yokota Hirai The proteinogenic amino acid l-serine is a precursor for various essential biomolecules in all organisms. 3-Phosphoglycerate dehydrogenase (PGDH) is the first committed enzyme of the phosphorylated pathway of l-serine biosynthesis, and is regulated by negative feedback from l-serine in bacteria and plants. In the present study, two Arabidopsis PGDH isoforms were inhibited by l-serine but were activated by l-amino acids such as l-homocysteine in vitro. Activation and inhibition by these amino acids was cooperative, suggesting an allosteric mechanism. Moreover, the half maximal effective concentration of l-homocysteine was 2 orders of magnitude lower than that of l-serine, suggesting greater regulatory potency. These are the first data to show that PGDH is activated by various biomolecules and indicate that serine biosynthesis is regulated by multiple pathways. In plants, l-serine is predominantly synthesized by glycolate and phosphorylated pathways. The glycolate path- way occurs in the mitochondria and is part of the photorespiratory pathway. This route is considered the main biosynthetic pathway of l-serine, at least in photosynthetic cells1, 2. The phosphorylated pathway (Fig. 1a), which is found in mammals, bacteria, and plants, comprises reactions of 3-phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase, and phosphoserine phosphatase2, and occurs in plastids3. The first committed enzyme PGDH oxidizes 3-phosphoglycerate (3-PGA), which originates from glycolysis and the Calvin cycle in heterotrophic and photosynthetic autotroph cells2, 4, 5.
    [Show full text]
  • An Integrated Study of Threonine-Pathway Enzyme Kinetics in Escherichia Coli Christophe CHASSAGNOLE*, Badr RAI$S*, Eric QUENTIN†1, David A
    Biochem. J. (2001) 356, 415–423 (Printed in Great Britain) 415 An integrated study of threonine-pathway enzyme kinetics in Escherichia coli Christophe CHASSAGNOLE*, Badr RAI$S*, Eric QUENTIN†1, David A. FELL*‡ and Jean-Pierre MAZAT*2 *INSERM EMI 9929, University Victor Segalen Bordeaux 2, 146 rue Le! o Saignat, 33076 Bordeaux, France, †De! partement de Biochimie Me! dicale et Biologie Mole! culaire, University Victor Segalen Bordeaux 2, 33076 Bordeaux, France, and ‡School of Biological and Molecular Sciences, Oxford Brookes University, Oxford OX3 0BP, U.K. We have determined the kinetic parameters of the individual the simplest rate equations that describe the salient features steps of the threonine pathway from aspartate in Escherichia coli of the enzymes in the physiological range of metabolite concen- under a single set of experimental conditions chosen to be trations in order to incorporate them ultimately into a complete physiologically relevant. Our aim was to summarize the kinetic model of the threonine pathway, able to predict quantitatively behaviour of each enzyme in a single tractable equation that the behaviour of the pathway under natural or engineered takes into account the effect of the products as competitive conditions. inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by Key words: amino acid, enzymology, model fitting, parameter threonine and lysine was also included as necessary. We derived estimation, rate equation. INTRODUCTION tensively, the complete genome sequence of this organism is now known and there is an extensive range of genetic tools available.
    [Show full text]