SYNTHESIS OF L-[ 15 N]-ISOLEUCINE, L-[ 15 N]-NORLEUCINE AND L-[ 15 N]-NORVALINE BY ENZYMATIC METHOD

Size: px
Start display at page:

Download "SYNTHESIS OF L-[ 15 N]-ISOLEUCINE, L-[ 15 N]-NORLEUCINE AND L-[ 15 N]-NORVALINE BY ENZYMATIC METHOD"

Transcription

1 SYNTHESIS OF L-[ 15 N]-ISOLEUCINE, L-[ 15 N]-NORLEUCINE AND L-[ 15 N]-NORVALINE BY ENZYMATIC METHOD IULIA LUPAN 1, BEATRIX FERENCZ 1, MARIA CHIRIAC 2, NADIA BUCURENCI 3, OCTAVIAN POPESCU 1 1 Molecular Biology Center, Institute for Interdisciplinary Experimental Research, Babeş-Bolyai University, Str. August Treboniu Laurian 42, Cluj-Napoca, Romania 2 National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, Romania 3 Cantacuzino National Institute of Research and Development for Microbiology and Immunology, Spl. Independenţei 103, Bucharest, Romania (Received September 15, 2006) Leucine dehydrogenase (Leu DH) (EC ) is a NAD-dependent oxidoreductase that catalyses reversible deamination of L-leucine and other aliphatic L-amino acids to their keto analogues. We cloned the structural gene (leudh) of Bacillus stearothermophilus into pet21b vector and expressed in E.coli strain BL21(DE3). The recombinant LeuDH was used for preparing L-[ 15 N]-isoleucine, L-[ 15 N]-norleucine and L-[ 15 N]-norvaline from the corresponding α-keto acids, by a simple procedure. We used a coupled enzymatic system: LeuDH as a catalyst and glucose dehydrogenase for NADH regeneration, using glucose as an electron donor. Reaction efficiencies were between 80 and 95%. Our results demonstrate the utility of recombinant amino acid dehydrogenases for synthesis of 15 N-labeled amino acids and the advantages of the enzymatic method. Key words: 15 N-labeled amino acid, leucine dehydrogenase, enzyme expression, Bacillus stearothermophilus. INTRODUCTION Stable isotope-labeled amino acids, particularly dual- or triple-labeled, can provide extremely high resolution structural information and form an important part of the toolkit for researchers in academia and industry, in a range of applications including: structural determinations of proteins, protein protein and protein drug interaction studies, metabolism and nutritional research, as well as labeled protein synthesis. 15 N-labeled amino acids are commonly commercially unavailable or expensive. The chemical synthesis of amino acids is disadvantageous, because a racemic product is produced. Differently, by the enzymatic method only one isomer (L or D) can be produced. Considerable attention has been paid to multienzyme reaction systems implied in stereospecific production of amino acids (1). ROM. J. BIOCHEM., 43 44, ( )

2 32 Iulia Lupan et al. 2 A method for enzymatic synthesis of L-amino acids from α-keto acids, using NAD(P)-dependent amino acid dehydrogenases, has been proposed (2, 3). Amino acid dehydrogenases are NAD-dependent oxidoreductases which catalyze the reversible deamination of L-amino acids to their keto analogues: L-amino acid + NAD + + H 2 O α-keto acid + NH NADH L-amino acids can be obtained in an almost quantitative manner by the reductive amination of keto acids, because the reaction equilibrium favors amino acid formation. Leucine dehydrogenase (LeuDH) (EC ) catalyzes the reversible deamination of L-leucine and other aliphatic L-amino acids to their keto analogues. The enzyme occurs ubiquitously in Bacillus species and functions catabolically in the bacterial metabolism of branched-chain L-amino acids. The enzyme has been purified to homogeneity from B. sphaericus (4), B. cereus (5), B. caldolyticus (6), and some thermophiles such as B. stearothermophilus (7), B. licheniformis (8), Clostridium thermoaceticum (9) and Thermoactinomyces intermedius (10). Several L-amino acids have been prepared using LeuDH in an enzyme-membrane reactor (11). MATERIAL AND METHODS Molecular biology methods. LeuDH gene from B. stearothermophilus was cloned in pet21b vector. The fragment for leudh gene was amplified by PCR, using the following primers: F1LDHBst 5 -GGTGGATCCGATGGAATTGTTCAAATATATGG-3 RLeBst 5 -TATTCTCGAGTATTGCCGAAGCACCTGC-3. The amplified fragment containing BamHI and XhoI linkers was digested and inserted in pet21b Novagen vector. The recombinant molecules were transformed into E. coli strain DH5α. The screening of the recombinant plasmids isolated from colonies obtained after transformation by electroporation was made by restriction enzyme digest. The cloned gene was sequenced with ABI Prism 310 Genetic Analyzer using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). In order to express the recombinant plasmid, the purified DNA was transferred into strain BL21(DE3). E. coli BL21(DE3) cells carrying recombinant plasmids were cultured at 37ºC in Luria-Bertani medium supplemented with 100 mg/l ampicillin. Biochemical methods. The standard reaction mixture for reductive amination consisted of 50 mm Tris-HCl ph 8.0, 10 mm α-keto acid, 0.15 mm NADH, 1 M NH 4 Cl and bacterial lysate with recombinant leucine dehydrogenase. The standard reaction mixture for glucose oxidation consists of 50 mm Tris-HCl ph 8.0, 10 mm glucose, 2 mm NAD and bacterial lysate with recombinant glucose dehydrogenase.

3 3 [ 15 N]-amino acid synthesis 33 One unit of enzyme was defined as the amount that catalyzed the formation (or consumption) of 1 µmol of NADH per minute under the standard assay conditions. When the coupled enzymatic system was used, the reaction mixture contained: 570 mm glucose, 115 mm keto acid, 115 mm NH 4 Cl (99% 15 N), 7 mm NADH. The excess of glucose was necessary for glucose dehydrogenase stability; it is known that the enzyme is unstable in the absence of glucose. The NH 4 Cl was assayed by the Berthelot method (12). The reaction mixture was stirred at 30 o C adjusting the ph value at 8 with 1N NaOH. Once no further change in ph was observed, the reaction mixture was heated at 85 o C for 15 min. After cooling, the denatured protein was removed by filtration or centrifugation and the reaction solution (50 ml) was loaded into a 120 ml column ( cm) of Dowex 50WX- 8 in H + form. After washing with distilled water, the adsorbed amino acid was eluted with 1 M NH 4 OH. The eluted fractions containing the labeled [ 15 N]-amino acid were concentrated in a rotary vacuum evaporator at 50 o C, to approximately 5 ml. Ninety ml of anhydrous ethanol/ether (50/50, v/v) were added and the mixture was kept overnight in the refrigerator, while the amino acid crystallized. The precipitate was filtered off and dried. RESULTS AND DISCUSSION The enzymatic method is the most simple and used technique to synthesize 15 N- labeled amino acids from keto acids and 15 NH 4 +. The advantage of the enzyme-catalyzed synthesis of amino acids by reductive amination is that it is restricted to the L-enantiomers as products. The corresponding NAD-depending D-amino acid dehydrogenases are not known. The ability of the recombinant leucine dehydrogenase obtained by us to catalyze the reductive amination of various keto acids is shown in Table 1. One can observe that α-ketoisovaleric acid is the preferred substrate. Table 1 Substrate specificities of recombinant leucine dehydrogenase in reductive amination α-keto acid Specific activity (U/mg protein) Relative activity (%) α-ketoisovaleric acid α-keto-β-methyl-n-valeric acid α-ketoisocaproic acid α-ketocaproic acid α-keto-γ-methylthiobutyric acid α-ketobutyric acid

4 34 Iulia Lupan et al. 4

5 5 [ 15 N]-amino acid synthesis 35 We synthesized L-[ 15 N]-isoleucine, L-[ 15 N]-norvaline and L-[ 15 N]-norleucine by reductive amination of DL-α-keto-β-methyl-n-valerate, α-ketovalerate and α-ketocaproate respectively in the presence of 15 NH 4 Cl and NADH and using recombinant LeuDH as a catalyst. As the NADH coenzyme is a very expensive and labile chemical, efficient coenzyme regeneration was needed to make this process economical. The simultaneous regeneration also offers a chance to overcome the stoichiometric relation between product and coenzyme by an efficient internal recycling. So, the reaction mixtures included in all cases glucose dehydrogenase and glucose for NADH regeneration. The ammonia consumption in L-[ 15 N]- isoleucine, L-[ 15 N]-norleucine and L-[ 15 N]-norvaline synthesis is given in Figure 1. Reaction efficiencies were between 80 and 95% (Table 2). Table 2 Reaction efficiencies of labeled amino acid enzymatic syntheses Amino acid Reaction efficiency (%) L-isoleucine 85 L-norleucine 80 L-norvaline 95 We show here the usefulness of recombinant leucine dehydrogenase and glucose dehydrogenase in enzymatic synthesis of 15 N-labelled amino acids. The NADH regeneration is more convenient with glucose dehydrogenase (the enzyme is more active and the substrates do not inhibit the reactions) than other methods described before with formate dehydrogenase (1) or alcohol dehydrogenase (13). Acknowledgements. This research was supported by the BIOTECH (3/260) and CNCSIS (code 1767) grants. REFERENCES 1. A. Galkin, L. Kulakova, T. Yoshimura, K. Soda, N. Esaki, Appl. Environ. Microbiol., 63, (1997). 2. T. Oshima, K. Soda, Int. Ind. Biotechnol., 9, 5 11 (1989). 3. A. Galkin, L. Kulakova, H. Yamamoto, K. Tanizawa, H.T. Tanaka, N. Esaki, K. Soda, J. Ferment. Bioeng., 83, (1997). 4. T. Ohshima, H. Misono, K Soda, J. Biol. Chem., 253, (1978). 5. H. Schutte, W. Hummel, H. Tsai, M.R. Kula, Appl. Microbiol. Biotechnol., 22, (1985). 6. U. Karst, H. Schutte, H. Baydoun, H. Tsai, J. Gen. Microbiol., 135, (1989). 7. T. Ohshima, S. Nagata, K. Soda, Arch. Microbiol., 141, (1985). 8. S. Nagata, S. Bakthavatsalam, A.G. Galkin, H. Asada, S. Sakai, N. Esaki, K. Soda, T. Ohshima, S. Nagasaki, K. Soda, Appl. Microbiol. Biotechnol., 44, (1995).

6 36 Iulia Lupan et al H. Shimoi, S. Nagata, N. Esaki, H. Tanaka, K. Soda, Agric. Biol. Chem., 51, (1987). 10. T. Ohshima, N. Nishida, S. Bakthavatsalam, K. Kataoka, H. Takada, T. Yoshimura, N. Esaki, K. Soda, Eur. J. Biochem., 222, (1994). 11. W. Hummel, M.R. Kula, Eur. J. Biochem., 184, 1-13 (1989). 12. M. Berthelot, Répertoire de Chemie appliqué, 1, 284 (1859). 13. A. Mocanu, G. Niac, A. Ivanof, V. Gorun, N. Palibroda, E. Varga, M. Bologa, O. Bârzu, FEBS Lett., 143, (1982).

Racemic Resolution of some DL-Amino Acids using Aspergillus fumigatus L-Amino Acid Oxidase

Racemic Resolution of some DL-Amino Acids using Aspergillus fumigatus L-Amino Acid Oxidase Curr Microbiol (2011) 63:94 99 DOI 10.1007/s00284-011-9955-8 Racemic Resolution of some DL-Amino Acids using Aspergillus fumigatus L-Amino Acid Oxidase Susmita Singh Binod K. Gogoi Rajib L. Bezbaruah Received:

More information

Distribution, Purification, and Characterization of Thermostable Phenylalanine Dehydrogenase from Thermophilic Actinomycetes

Distribution, Purification, and Characterization of Thermostable Phenylalanine Dehydrogenase from Thermophilic Actinomycetes JOURNAL OF BACTERIOLOGY, JUlY 1991, p. 3943-3948 Vol. 173, No. 13 0021-9193/91/133943-06$02.00/0 Copyright 1991, American Society for Microbiology Distribution, Purification, and Characterization of Thermostable

More information

BCH 447. Estimation of Serum Urea

BCH 447. Estimation of Serum Urea BCH 447 Estimation of Serum Urea 1 Objective: Estimation of Blood urea nitrogen (BUN) in serum sample. 2 -Urea: Urea is the highest non-protein nitrogen compound in the blood. Urea is the major excretory

More information

Marah Bitar. Faisal Nimri ... Nafeth Abu Tarboosh

Marah Bitar. Faisal Nimri ... Nafeth Abu Tarboosh 8 Marah Bitar Faisal Nimri... Nafeth Abu Tarboosh Summary of the 8 steps of citric acid cycle Step 1. Acetyl CoA joins with a four-carbon molecule, oxaloacetate, releasing the CoA group and forming a six-carbon

More information

Metabolic engineering some basic considerations. Lecture 9

Metabolic engineering some basic considerations. Lecture 9 Metabolic engineering some basic considerations Lecture 9 The 90ties: From fermentation to metabolic engineering Recruiting heterologous activities to perform directed genetic modifications of cell factories

More information

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2008

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2008 Experimental Details Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich Chemical Company and were used as received. 2-DOS and neamine were kindly provided by Dr. F. Huang. Paromamine

More information

Foundations in Microbiology Seventh Edition

Foundations in Microbiology Seventh Edition Lecture PowerPoint to accompany Foundations in Microbiology Seventh Edition Talaro Chapter 8 An Introduction to Microbial Metabolism Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction

More information

KE-SIALIQ Sialic Acid Quantitation Kit. SialiQuant Sialic Acid Quantitation Kit

KE-SIALIQ Sialic Acid Quantitation Kit. SialiQuant Sialic Acid Quantitation Kit SialiQuant Sialic Acid Quantitation Kit Part Number KE-SIALIQ Certification of Analysis Lot Number 706.1A Kit Storage Kits should be stored at 4 C. Kit Contents Kit contains all the reagents to quickly

More information

Metabolism Energy Pathways Biosynthesis. Catabolism Anabolism Enzymes

Metabolism Energy Pathways Biosynthesis. Catabolism Anabolism Enzymes Topics Microbial Metabolism Metabolism Energy Pathways Biosynthesis 2 Metabolism Catabolism Catabolism Anabolism Enzymes Breakdown of complex organic molecules in order to extract energy and dform simpler

More information

Respiration. Respiration. Respiration. How Cells Harvest Energy. Chapter 7

Respiration. Respiration. Respiration. How Cells Harvest Energy. Chapter 7 How Cells Harvest Energy Chapter 7 Organisms can be classified based on how they obtain energy: autotrophs: are able to produce their own organic molecules through photosynthesis heterotrophs: live on

More information

Biochemistry: A Short Course

Biochemistry: A Short Course Tymoczko Berg Stryer Biochemistry: A Short Course Second Edition CHAPTER 30 Amino Acid Degradation and the Urea Cycle 2013 W. H. Freeman and Company Chapter 30 Outline Amino acids are obtained from the

More information

Student Handout. This experiment allows you to explore the properties of chiral molecules. You have

Student Handout. This experiment allows you to explore the properties of chiral molecules. You have Student Handout This experiment allows you to explore the properties of chiral molecules. You have learned that some compounds exist as enantiomers non-identical mirror images, such as your left and right

More information

GLYCATION OF PROTEINS IN ESCHERICHIA COLI: EFFECT OF NUTRIENT BROTH INGREDIENTS ON GLYCATION

GLYCATION OF PROTEINS IN ESCHERICHIA COLI: EFFECT OF NUTRIENT BROTH INGREDIENTS ON GLYCATION Industry GLYCATION OF PROTEINS IN ESCHERICHIA COLI: EFFECT OF NUTRIENT BROTH INGREDIENTS ON GLYCATION R. Dimitrova, R. Mironova, I. Ivanov Institute of Molecular biology, Bulgarian Academy of Sciences

More information

Ch 07. Microbial Metabolism

Ch 07. Microbial Metabolism Ch 07 Microbial Metabolism SLOs Differentiate between metabolism, catabolism, and anabolism. Fully describe the structure and function of enzymes. Differentiate between constitutive and regulated enzymes.

More information

Cellular Pathways That Harvest Chemical Energy. Cellular Pathways That Harvest Chemical Energy. Cellular Pathways In General

Cellular Pathways That Harvest Chemical Energy. Cellular Pathways That Harvest Chemical Energy. Cellular Pathways In General Cellular Pathways That Harvest Chemical Energy A. Obtaining Energy and Electrons from Glucose Lecture Series 12 Cellular Pathways That Harvest Chemical Energy B. An Overview: Releasing Energy from Glucose

More information

NOTES: Ch 9 Cellular Respiration: Harvesting Chemical Energy Part 1: The Overview

NOTES: Ch 9 Cellular Respiration: Harvesting Chemical Energy Part 1: The Overview NOTES: Ch 9 Cellular Respiration: Harvesting Chemical Energy Part 1: The Overview Overview: Life Is Work Living cells require energy from outside sources Some animals, such as the giant panda, obtain energy

More information

Physiology 12. Metabolism. Metabolism. Cellular metabolism. The synthesis and Breakdown of organic molecules required for cell structure and function

Physiology 12. Metabolism. Metabolism. Cellular metabolism. The synthesis and Breakdown of organic molecules required for cell structure and function Physiology 12 Cellular metabolism Germann Ch3 Metabolism The synthesis and Breakdown of organic molecules required for cell structure and function Metabolism Anabolism = Synthesis Catabolism = Breaking

More information

How Cells Harvest Energy. Chapter 7. Respiration

How Cells Harvest Energy. Chapter 7. Respiration How Cells Harvest Energy Chapter 7 Respiration Organisms classified on how they obtain energy: autotrophs: produce their own organic molecules through photosynthesis heterotrophs: live on organic compounds

More information

SPRIN Protease Kit. Content Code Description Application. Covalently immobilised preparation of Subtilisin. Epoxy Acrylic Resin

SPRIN Protease Kit. Content Code Description Application. Covalently immobilised preparation of Subtilisin. Epoxy Acrylic Resin SPRIN Protease Kit SPRIN Protease Kit Normal (5 g each) Product code: SPKN SPRIN Protease Kit Large (20 g each) Product code: SPKL The kit contains four different covalently immobilised protease preparations,

More information

Estimation of Serum Urea

Estimation of Serum Urea Estimation of Serum Urea 1 -Urea: Urea is the highest non-protein nitrogen compound in the blood. Urea is the major excretory product of protein metabolism. It is formed by urea cycle in the liver from

More information

Supporting information (protein purification, kinetic characterization, product isolation, and characterization by NMR and mass spectrometry):

Supporting information (protein purification, kinetic characterization, product isolation, and characterization by NMR and mass spectrometry): Supporting Information Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate Heng Song, 1 Wen Hu, 1,2 Nathchar Naowarojna, 1 Ampon Sae

More information

TECHNICAL BULLETIN. Sialic Acid Quantitation Kit. Catalog Number SIALICQ Storage Temperature 2 8 C

TECHNICAL BULLETIN. Sialic Acid Quantitation Kit. Catalog Number SIALICQ Storage Temperature 2 8 C Sialic Acid Quantitation Kit Catalog Number SIALICQ Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description The Sialic Acid Quantitation Kit provides a rapid and accurate determination of total

More information

Caution: For Laboratory Use. A product for research purposes only. Eu-W1024 ITC Chelate & Europium Standard. Product Number: AD0013

Caution: For Laboratory Use. A product for research purposes only. Eu-W1024 ITC Chelate & Europium Standard. Product Number: AD0013 TECHNICAL DATA SHEET Lance Caution: For Laboratory Use. A product for research purposes only. Eu-W1024 ITC Chelate & Europium Standard Product Number: AD0013 INTRODUCTION: Fluorescent isothiocyanato-activated

More information

Coenzymes, vitamins and trace elements 209. Petr Tůma Eva Samcová

Coenzymes, vitamins and trace elements 209. Petr Tůma Eva Samcová Coenzymes, vitamins and trace elements 209 Petr Tůma Eva Samcová History and nomenclature of enzymes 1810, Gay-Lussac made an experiment with yeats alter saccharide to ethanol and CO 2 Fermentation From

More information

Organic and biochemical synthesis of monolignol biosynthetic pathway intermediates

Organic and biochemical synthesis of monolignol biosynthetic pathway intermediates Jie Liu 2012-2-8 Organic and biochemical synthesis of monolignol biosynthetic pathway intermediates 1. Organic synthesis of 5-hydroxyferulic acid Malonic acid 3, 4-Dihydroxy-5-methoxy-benzaldehyde 0.1

More information

LANCE Eu-W1024 ITC Chelate & Europium Standard AD0013 Development grade

LANCE Eu-W1024 ITC Chelate & Europium Standard AD0013 Development grade AD0017P-4 (en) 1 LANCE Eu-W1024 ITC Chelate & Europium Standard AD0013 Development grade INTRODUCTION Fluorescent isothiocyanato-activated (ITC-activated) Eu-W1024 chelate is optimized for labelling proteins

More information

Experiment 1. Isolation of Glycogen from rat Liver

Experiment 1. Isolation of Glycogen from rat Liver Experiment 1 Isolation of Glycogen from rat Liver Figure 35: FIG-2, Liver, PAS, 100x. Note the presence of a few scattered glycogen granules (GG). Objective To illustrate the method for isolating glycogen.

More information

Concept 8.3: ATP powers cellular work by coupling exergonic reactions to endergonic reactions

Concept 8.3: ATP powers cellular work by coupling exergonic reactions to endergonic reactions Concept 8.3: ATP powers cellular work by coupling exergonic reactions to endergonic reactions A cell does three main kinds of work: Chemical Transport Mechanical To do work, cells manage energy resources

More information

Novozymes Protease Products. Strem Chemicals, Inc. Storage

Novozymes Protease Products. Strem Chemicals, Inc. Storage Strem Chemicals, Inc. www.strem.com Catalog # 06-3160 Neutrase 0.8 L Note: Sold in collaboration with Novozymes A/S for research purposes only. Neutrase 0.8 L is a neutral, zinc metallo endo-protease from

More information

Synthesis and Blastocyst Implantation Inhibition Potential of Lupeol Derivatives in Female Mice

Synthesis and Blastocyst Implantation Inhibition Potential of Lupeol Derivatives in Female Mice Supporting Information Rec. Nat. Prod. 9:4 (2015) 561-566 Synthesis and Blastocyst Implantation Inhibition Potential of Lupeol Derivatives in Female Mice Anita Mahapatra 1*, Purvi Shah 1, Mehul Jivrajani

More information

Biochemistry: A Short Course

Biochemistry: A Short Course Tymoczko Berg Stryer Biochemistry: A Short Course Second Edition CHAPTER 30 Amino Acid Degradation and the Urea Cycle 2013 W. H. Freeman and Company In the cytosol of a cell amino groups from amino acids

More information

Amino acid metabolism

Amino acid metabolism Amino acid metabolism The important reaction commonly employed in the breakdown of an amino acid is always the removal of its -amino group. The product ammonia is excreted after conversion to urea or other

More information

Caution: For Laboratory Use. A product for research purposes only. Eu-W1284 Iodoacetamido Chelate & Europium Standard. Product Number: AD0014

Caution: For Laboratory Use. A product for research purposes only. Eu-W1284 Iodoacetamido Chelate & Europium Standard. Product Number: AD0014 TECHNICAL DATA SHEET Lance Caution: For Laboratory Use. A product for research purposes only. Eu-W1284 Iodoacetamido Chelate & Europium Standard Product Number: AD0014 INTRODUCTION: Iodoacetamido-activated

More information

Two Types of Inulin Fructotransferases

Two Types of Inulin Fructotransferases Materials 2011, 4, 1543-1547; doi:10.3390/ma4091543 Short Note OPEN ACCESS materials ISSN 1996-1944 www.mdpi.com/journal/materials Two Types of Inulin Fructotransferases Kazutomo Haraguchi National Food

More information

Energy and life. Generation of Biochemical Energy Chapter 21. Energy. Energy and biochemical reactions: 4/5/09

Energy and life. Generation of Biochemical Energy Chapter 21. Energy. Energy and biochemical reactions: 4/5/09 Energy and life Generation of Biochemical Energy Chapter 21 1 Biological systems are powered by oxidation of biomolecules made mainly of C, H and O. The food biomolecules are mainly Lipids (fats) Carbohydrates

More information

Supporting Information. Copper-catalyzed cascade synthesis of benzimidazoquinazoline derivatives under mild condition

Supporting Information. Copper-catalyzed cascade synthesis of benzimidazoquinazoline derivatives under mild condition Supporting Information Copper-catalyzed cascade synthesis of benzimidazoquinazoline derivatives under mild condition Shan Xu, Juyou Lu and Hua Fu* Key Laboratory of Bioorganic Phosphorus Chemistry and

More information

Respiration. Respiration. How Cells Harvest Energy. Chapter 7

Respiration. Respiration. How Cells Harvest Energy. Chapter 7 How Cells Harvest Energy Chapter 7 Respiration Organisms can be classified based on how they obtain energy: autotrophs: are able to produce their own organic molecules through photosynthesis heterotrophs:

More information

Supporting Information for:

Supporting Information for: Supporting Information for: Methylerythritol Cyclodiphosphate (MEcPP) in Deoxyxylulose Phosphate Pathway: Synthesis from an Epoxide and Mechanisms Youli Xiao, a Rodney L. Nyland II, b Caren L. Freel Meyers

More information

MULTIPLE CHOICE QUESTIONS

MULTIPLE CHOICE QUESTIONS MULTIPLE CHOICE QUESTIONS 1. Which of the following statements concerning anabolic reactions is FALSE? A. They are generally endergonic. B. They usually require ATP. C. They are part of metabolism. D.

More information

Chemical Energy. Valencia College

Chemical Energy. Valencia College 9 Pathways that Harvest Chemical Energy Valencia College 9 Pathways that Harvest Chemical Energy Chapter objectives: How Does Glucose Oxidation Release Chemical Energy? What Are the Aerobic Pathways of

More information

Supporting Information. were prepared from commercially available ethyl acetoacetate by alkylation with the

Supporting Information. were prepared from commercially available ethyl acetoacetate by alkylation with the ighly Stereoselective Reductions of α-alkyl-1,3-diketones and α- Alkyl-β-keto esters Catalyzed by Isolated NADP-dependent Ketoreductases Dimitris Kalaitzakis, a David J. Rozzell b, Spiros Kambourakis *b

More information

NITROGEN METABOLISM An Overview

NITROGEN METABOLISM An Overview 1 University of Papua New Guinea School of Medicine and Health Sciences Division of Basic Medical Sciences Discipline of Biochemistry and Molecular Biology PBL Seminar & Health Sciences NITROGEN METABOLISM

More information

Concept 9.1: Catabolic pathways yield energy by oxidizing organic fuels Several processes are central to cellular respiration and related pathways

Concept 9.1: Catabolic pathways yield energy by oxidizing organic fuels Several processes are central to cellular respiration and related pathways Overview: Life Is Work Living cells require energy from outside sources Some animals, such as the chimpanzee, obtain energy by eating plants, and some animals feed on other organisms that eat plants Energy

More information

Energy Production In A Cell (Chapter 25 Metabolism)

Energy Production In A Cell (Chapter 25 Metabolism) Energy Production In A Cell (Chapter 25 Metabolism) Large food molecules contain a lot of potential energy in the form of chemical bonds but it requires a lot of work to liberate the energy. Cells need

More information

The Conservation of Homochirality and Prebiotic Synthesis of Amino Acids

The Conservation of Homochirality and Prebiotic Synthesis of Amino Acids The Conservation of Homochirality and Prebiotic Synthesis of Amino Acids Harold J. Morowitz SFI WORKING PAPER: 2001-03-017 SFI Working Papers contain accounts of scientific work of the author(s) and do

More information

Luminescent platforms for monitoring changes in the solubility of amylin and huntingtin in living cells

Luminescent platforms for monitoring changes in the solubility of amylin and huntingtin in living cells Electronic Supplementary Material (ESI) for Molecular BioSystems. This journal is The Royal Society of Chemistry 2016 Contents Supporting Information Luminescent platforms for monitoring changes in the

More information

Chapter 9. Cellular Respiration and Fermentation

Chapter 9. Cellular Respiration and Fermentation Chapter 9 Cellular Respiration and Fermentation Energy flows into an ecosystem as sunlight and leaves as heat Photosynthesis generates O 2 and organic molecules, which are used in cellular respiration

More information

Conversion of green note aldehydes into alcohols by yeast alcohol dehydrogenase

Conversion of green note aldehydes into alcohols by yeast alcohol dehydrogenase Conversion of green note aldehydes into alcohols by yeast alcohol dehydrogenase M.-L. Fauconnier 1, A. Mpambara 1, J. Delcarte 1, P. Jacques 2, P. Thonart 2 & M. Marlier 1 1 Unité de Chimie Générale et

More information

AMINO ACIDS NON-ESSENTIAL ESSENTIAL

AMINO ACIDS NON-ESSENTIAL ESSENTIAL Edith Frederika Introduction A major component of food is PROTEIN The protein ingested as part of our diet are not the same protein required by the body Only 40 to 50 gr of protein is required by a normal

More information

BIOLOGY - CLUTCH CH.9 - RESPIRATION.

BIOLOGY - CLUTCH CH.9 - RESPIRATION. !! www.clutchprep.com CONCEPT: REDOX REACTIONS Redox reaction a chemical reaction that involves the transfer of electrons from one atom to another Oxidation loss of electrons Reduction gain of electrons

More information

Construction of a hepatocellular carcinoma cell line that stably expresses stathmin with a Ser25 phosphorylation site mutation

Construction of a hepatocellular carcinoma cell line that stably expresses stathmin with a Ser25 phosphorylation site mutation Construction of a hepatocellular carcinoma cell line that stably expresses stathmin with a Ser25 phosphorylation site mutation J. Du 1, Z.H. Tao 2, J. Li 2, Y.K. Liu 3 and L. Gan 2 1 Department of Chemistry,

More information

Amino acid Catabolism

Amino acid Catabolism Enzymatic digestion of dietary proteins in gastrointestinal-tract. Amino acid Catabolism Amino acids: 1. There are 20 different amino acid, they are monomeric constituents of proteins 2. They act as precursors

More information

Volatile Fatty Acids and the Inhibition of Escherichia

Volatile Fatty Acids and the Inhibition of Escherichia APPuan MICROBIOLOGY, Jan. 1969, p. 83-87 Copyright 1969 American Society for Microbiology Vol. 17, No. 1 Printed in U.S.A Volatile Fatty Acids and the of Escherichia coli Growth by Rumen Fluid1 MEYER J.

More information

III. Metabolism The Citric Acid Cycle

III. Metabolism The Citric Acid Cycle Department of Chemistry and Biochemistry University of Lethbridge III. Metabolism The Citric Acid Cycle Slide 1 The Eight Steps of the Citric Acid Cycle Enzymes: 4 dehydrogenases (2 decarboxylation) 3

More information

2/25/2013. The Mechanism of Enzymatic Action

2/25/2013. The Mechanism of Enzymatic Action 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Chapter 5 Microbial Metabolism Catabolic and Anabolic Reactions Metabolism: The sum of the chemical reactions in an organism Catabolic and Anabolic Reactions Catabolism:

More information

Reading Assignments. A. Energy and Energy Conversions. Lecture Series 9 Cellular Pathways That Harvest Chemical Energy. gasoline) or elevated mass.

Reading Assignments. A. Energy and Energy Conversions. Lecture Series 9 Cellular Pathways That Harvest Chemical Energy. gasoline) or elevated mass. Lecture Series 9 Cellular Pathways That Harvest Chemical Energy Reading Assignments Review Chapter 3 Energy, Catalysis, & Biosynthesis Read Chapter 13 How Cells obtain Energy from Food Read Chapter 14

More information

CHAPTER 5 MICROBIAL METABOLISM

CHAPTER 5 MICROBIAL METABOLISM CHAPTER 5 MICROBIAL METABOLISM I. Catabolic and Anabolic Reactions A. Metabolism - The sum of all chemical reactions within a living cell either releasing or requiring energy. (Overhead) Fig 5.1 1. Catabolism

More information

Amino acid metabolism I

Amino acid metabolism I Amino acid metabolism I Jana Novotná Department of the Medical Chemistry and Clinical Biochemistry The 2nd Faculty of Medicine, Charles Univ. Metabolic relationship of amino acids DIETARY PROTEINS GLYCOLYSIS

More information

Metabolism of proteins and amino acids

Metabolism of proteins and amino acids BIOQUÍMICA E BIOLOGIA CELULAR António Ascensão, José Magalhães Metabolism of proteins and amino acids Faculdade de Desporto, Universidade do Porto, 1º Ciclo, 1º Ano 202_2013 Humans degradation of ingested

More information

BASIC ENZYMOLOGY 1.1

BASIC ENZYMOLOGY 1.1 BASIC ENZYMOLOGY 1.1 1.2 BASIC ENZYMOLOGY INTRODUCTION Enzymes are synthesized by all living organisms including man. These life essential substances accelerate the numerous metabolic reactions upon which

More information

7 Pathways That Harvest Chemical Energy

7 Pathways That Harvest Chemical Energy 7 Pathways That Harvest Chemical Energy Pathways That Harvest Chemical Energy How Does Glucose Oxidation Release Chemical Energy? What Are the Aerobic Pathways of Glucose Metabolism? How Is Energy Harvested

More information

Microbial Metabolism. PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College C H A P T E R

Microbial Metabolism. PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College C H A P T E R PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College C H A P T E R 5 Microbial Metabolism Big Picture: Metabolism Metabolism is the buildup and breakdown of nutrients

More information

Metabolism. Chapter 8 Microbial Metabolism. Metabolic balancing act. Catabolism Anabolism Enzymes. Topics. Metabolism Energy Pathways Biosynthesis

Metabolism. Chapter 8 Microbial Metabolism. Metabolic balancing act. Catabolism Anabolism Enzymes. Topics. Metabolism Energy Pathways Biosynthesis Chapter 8 Microbial Metabolism Topics Metabolism Energy Pathways Biosynthesis Catabolism Anabolism Enzymes Metabolism 1 2 Metabolic balancing act Catabolism and anabolism simple model Catabolism Enzymes

More information

Conversion of a-amino Acids into Nitriles by Oxidative Decarboxylation with Trichloroisocyanuric Acid

Conversion of a-amino Acids into Nitriles by Oxidative Decarboxylation with Trichloroisocyanuric Acid SYNTHETIC COMMUNICATIONS w Vol. 34, No. 19, pp. 3449 3453, 2004 Conversion of a-amino Acids into Nitriles by Oxidative Decarboxylation with Trichloroisocyanuric Acid Gene A. Hiegel,* Justin C. Lewis, and

More information

Independent Study Guide Metabolism I. Principles of metabolism (section 6.1) a. Cells must: (figure 6.1) i. Synthesize new components

Independent Study Guide Metabolism I. Principles of metabolism (section 6.1) a. Cells must: (figure 6.1) i. Synthesize new components Independent Study Guide Metabolism I. Principles of metabolism (section 6.1) a. Cells must: (figure 6.1) i. Synthesize new components (anabolism/biosynthesis) ii. Harvest energy and convert it to a usable

More information

Chemicals Based on Ethylene

Chemicals Based on Ethylene Chemicals Based on Ethylene Ethylene is sometimes known as the king of petrochemicals because more commercial chemicals are produced from ethylene than from any other intermediate. This unique position

More information

Chem Lecture 8 Carbohydrate Metabolism Part I: Glycolysis

Chem Lecture 8 Carbohydrate Metabolism Part I: Glycolysis Chem 352 - Lecture 8 Carbohydrate Metabolism Part I: Glycolysis Introduction Carbohydrate metabolism involves a collection of pathways. Glycolysis Hexoses 3-Carbon molecules Gluconeogenesis 3-Carbon molecules

More information

Catabolism of Carbon skeletons of Amino acids. Amino acid metabolism

Catabolism of Carbon skeletons of Amino acids. Amino acid metabolism Catabolism of Carbon skeletons of Amino acids Amino acid metabolism Carbon skeleton Carbon Skeleton a carbon skeleton is the internal structure of organic molecules. Carbon Arrangements The arrangement

More information

Citric Acid Cycle: Central Role in Catabolism. Entry of Pyruvate into the TCA cycle

Citric Acid Cycle: Central Role in Catabolism. Entry of Pyruvate into the TCA cycle Citric Acid Cycle: Central Role in Catabolism Stage II of catabolism involves the conversion of carbohydrates, fats and aminoacids into acetylcoa In aerobic organisms, citric acid cycle makes up the final

More information

Six Types of Enzyme Catalysts

Six Types of Enzyme Catalysts Six Types of Enzyme Catalysts Although a huge number of reactions occur in living systems, these reactions fall into only half a dozen types. The reactions are: 1. Oxidation and reduction. Enzymes that

More information

EFFECT OF SOME AMINO ACIDS ON THE GROWTH AND L-GLUTAMIC ACID FERMENTATION BY AN AUXOTROPHIC MUTANT Micrococcus glutamicus AB 100.

EFFECT OF SOME AMINO ACIDS ON THE GROWTH AND L-GLUTAMIC ACID FERMENTATION BY AN AUXOTROPHIC MUTANT Micrococcus glutamicus AB 100. S. Ganguly et. al. / International Journal on Pharmaceutical and Biomedical Research (IJPBR) Vol. 2(1), 2011, 21-25 EFFECT OF SOME AMINO ACIDS ON THE GROWTH AND L-GLUTAMIC ACID FERMENTATION BY AN AUXOTROPHIC

More information

Case 19 Purification of Rat Kidney Sphingosine Kinase

Case 19 Purification of Rat Kidney Sphingosine Kinase Case 19 Purification of Rat Kidney Sphingosine Kinase Focus concept The purification and kinetic analysis of an enzyme that produces a product important in cell survival is the focus of this study. Prerequisites

More information

Chapter 8. Metabolism. Topics in lectures 15 and 16. Chemical foundations Catabolism Biosynthesis

Chapter 8. Metabolism. Topics in lectures 15 and 16. Chemical foundations Catabolism Biosynthesis Chapter 8 Topics in lectures 15 and 16 Metabolism Chemical foundations Catabolism Biosynthesis 1 Metabolism Chemical Foundations Enzymes REDOX Catabolism Pathways Anabolism Principles and pathways 2 Enzymes

More information

SOIL ENZYMES. Tento projekt je spolufinancován Evropským sociálním fondem a Státním rozpočtem ČR InoBio CZ.1.07/2.2.00/

SOIL ENZYMES. Tento projekt je spolufinancován Evropským sociálním fondem a Státním rozpočtem ČR InoBio CZ.1.07/2.2.00/ Soil Biology topic No. 11: SOIL ENZYMES Enzymes Simple (only proteins) Composite protein + cofactor coenzyme (NAD +, NADP +, FAD +,FADP +, hem, α lipoate, Fe ions etc.) = holoenzyme) prosthetic group (coenzyme

More information

Student Number: THE UNIVERSITY OF MANITOBA April 11, 2011, 1:00 PM - 4:00 PM Page 1 (of 3)

Student Number: THE UNIVERSITY OF MANITOBA April 11, 2011, 1:00 PM - 4:00 PM Page 1 (of 3) Name: Student Number: THE UNIVERSITY OF MANITOBA April 11, 2011, 1:00 PM - 4:00 PM Page 1 (of 3) Biochemistry II Laboratory Section Examiners: Drs. J. Galka 1. Answer ALL questions in the space provided.

More information

Chapter 9. Cellular Respiration: Harvesting Chemical Energy

Chapter 9. Cellular Respiration: Harvesting Chemical Energy Chapter 9 Cellular Respiration: Harvesting Chemical Energy Living cells require energy from outside sources Energy flows into an ecosystem as sunlight and leaves as heat Photosynthesis generates O 2 and

More information

Citrate Cycle. Lecture 28. Key Concepts. The Citrate Cycle captures energy using redox reactions

Citrate Cycle. Lecture 28. Key Concepts. The Citrate Cycle captures energy using redox reactions Citrate Cycle Lecture 28 Key Concepts The Citrate Cycle captures energy using redox reactions Eight reactions of the Citrate Cycle Key control points in the Citrate Cycle regulate metabolic flux What role

More information

Communication. Identification of Methionine N -Acetyltransferase from Saccharomyces cerevisiae

Communication. Identification of Methionine N -Acetyltransferase from Saccharomyces cerevisiae Communication THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 265, No. 7, Issue of March 5, pp. 3603-3606,lSSO 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A. Identification

More information

Foundations in Microbiology Seventh Edition

Foundations in Microbiology Seventh Edition Lecture PowerPoint to accompany Foundations in Microbiology Seventh Edition Talaro Chapter 8 To run the animations you must be in Slideshow View. Use the buttons on the animation to play, pause, and turn

More information

Branched Chain Amino Acid (Leu, Ile, Val) Assay Kit

Branched Chain Amino Acid (Leu, Ile, Val) Assay Kit Branched Chain Amino Acid (Leu, Ile, Val) Assay Kit Catalog Number KA0824 100 assays Version: 03 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General

More information

Student Number: To form the polar phase when adsorption chromatography was used.

Student Number: To form the polar phase when adsorption chromatography was used. Name: Student Number: April 14, 2001, 1:30 AM - 4:30 PM Page 1 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot 1. Answer ALL questions in the space provided.. 2. The last page

More information

Ketoreductase (KRED) FAQ

Ketoreductase (KRED) FAQ Tools for Feasibility Assessment and Accelerated Evolution page 1 of 6 Should I Use the Screening Kit or the Panel? Frequently Asked Questions KRED Screening Kit advantages over the KRED Panel: High chance

More information

Protein & Enzyme Lab (BBT 314)

Protein & Enzyme Lab (BBT 314) Protein & Enzyme Lab (BBT 314) Experiment 3 A: Determination of the enzyme ALT or SGPT activity in serum by enzymatic method using Bioanalyzer Background: Alanine aminotransferase (glutamate pyruvate transaminase)

More information

III. 6. Test. Respiració cel lular

III. 6. Test. Respiració cel lular III. 6. Test. Respiració cel lular Chapter Questions 1) What is the term for metabolic pathways that release stored energy by breaking down complex molecules? A) anabolic pathways B) catabolic pathways

More information

A biocatalytic hydrogenation of carboxylic acids

A biocatalytic hydrogenation of carboxylic acids Electronic Supplementary Information (ESI) for: A biocatalytic hydrogenation of carboxylic acids Yan Ni, Peter-Leon Hagedoorn,* Jian-He Xu, Isabel Arends, Frank Hollmann* 1. General Chemicals All the carboxylic

More information

Chapter 5 Microbial Metabolism: The Chemical Crossroads of Life

Chapter 5 Microbial Metabolism: The Chemical Crossroads of Life Chapter 5 Microbial Metabolism: The Chemical Crossroads of Life Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The Metabolism of Microbes metabolism all chemical

More information

Lecture 11 - Biosynthesis of Amino Acids

Lecture 11 - Biosynthesis of Amino Acids Lecture 11 - Biosynthesis of Amino Acids Chem 454: Regulatory Mechanisms in Biochemistry University of Wisconsin-Eau Claire 1 Introduction Biosynthetic pathways for amino acids, nucleotides and lipids

More information

Supplementary figure legends

Supplementary figure legends Supplementary figure legends Fig. S1. Lineweaver-Burk plot of putrescine uptake by YeeF. An overnight culture of SK629 was inoculated in 100-mL LBG medium in 500-mL Erlenmeyer flasks. The medium was supplemented

More information

Microbiology AN INTRODUCTION

Microbiology AN INTRODUCTION TORTORA FUNKE CASE Microbiology AN INTRODUCTION EIGHTH EDITION B.E Pruitt & Jane J. Stein Chapter 5, part A Microbial Metabolism PowerPoint Lecture Slide Presentation prepared by Christine L. Case Microbial

More information

BY: RASAQ NURUDEEN OLAJIDE

BY: RASAQ NURUDEEN OLAJIDE BY: RASAQ NURUDEEN OLAJIDE LECTURE CONTENT INTRODUCTION CITRIC ACID CYCLE (T.C.A) PRODUCTION OF ACETYL CoA REACTIONS OF THE CITIRC ACID CYCLE THE AMPHIBOLIC NATURE OF THE T.C.A CYCLE THE GLYOXYLATE CYCLE

More information

Chapter 8 Mitochondria and Cellular Respiration

Chapter 8 Mitochondria and Cellular Respiration Chapter 8 Mitochondria and Cellular Respiration Cellular respiration is the process of oxidizing food molecules, like glucose, to carbon dioxide and water. The energy released is trapped in the form of

More information

Supporting Information

Supporting Information Supporting Information Synthesis of N-Heteropolycyclic Compounds Including Quinazolinone Skeletons by Using Friedel-Crafts Alkylation Bu Keun Oh, Eun Bi Ko, Jin Wook Han* and Chang Ho Oh* Department of

More information

Electronic Supplementary Information

Electronic Supplementary Information Electronic Supplementary Information A Novel and Facile Zn-mediated Intramolecular Five-membered Cyclization of β-tetraarylporphyrin Radicals from β-bromotetraarylporphyrins Dong-Mei Shen, Chao Liu, Qing-Yun

More information

Effect of 6-Aminonicotinamide on the activity of hexokinase and lactate dehydrogenase isoenzymes in regions of the rat brain

Effect of 6-Aminonicotinamide on the activity of hexokinase and lactate dehydrogenase isoenzymes in regions of the rat brain J. Biosci., Vol. 6, Number 3, September 1984, pp. 331-336. Printed in India. Effect of 6-Aminonicotinamide on the activity of hexokinase and lactate dehydrogenase isoenzymes in regions of the rat brain

More information

Cellular Respiration Harvesting Chemical Energy ATP

Cellular Respiration Harvesting Chemical Energy ATP Cellular Respiration Harvesting Chemical Energy ATP 2006-2007 What s the point? The point is to make ATP! ATP 2006-2007 Harvesting stored energy Energy is stored in organic molecules carbohydrates, fats,

More information

Chem 454, Spring Exam I R, the ideal gas law constant = x 10-3 kj/mol = x 10-3 kcal/mol

Chem 454, Spring Exam I R, the ideal gas law constant = x 10-3 kj/mol = x 10-3 kcal/mol Name Chem 454, Spring 2003 - Exam I R, the ideal gas law constant = 8.314 x 10-3 kj/mol = 1.987 x 10-3 kcal/mol 1. Metabolism can be divided into two processes: catabolism and anabolism. a. Define metabolism,

More information

Cellular Respiration

Cellular Respiration Cellular I can describe cellular respiration Cellular respiration is a series of metabolic pathways releasing energy from a foodstuff e.g. glucose. This yields energy in the form of ATP adenosine P i P

More information

Enzymes what are they?

Enzymes what are they? Topic 11 (ch8) Microbial Metabolism Topics Metabolism Energy Pathways Biosynthesis 1 Catabolism Anabolism Enzymes Metabolism 2 Metabolic balancing act Catabolism Enzymes involved in breakdown of complex

More information

XXVI. STUDIES ON THE INTERACTION. OF AMINO-COMPOUNDS AND CARBOHYDRATES.

XXVI. STUDIES ON THE INTERACTION. OF AMINO-COMPOUNDS AND CARBOHYDRATES. XXVI. STUDIES ON THE INTERACTION. OF AMINO-COMPOUNDS AND CARBOHYDRATES. II. THE PREPARATION OF GLUCOSE UREIDE. BY ALEXANDER HYND. From the Department of Physiology, University of St Andrews. (Received

More information

Branched Chain Amino Acid, Leucine: The Effects of Leucine on Skeletal Tissue in Relation to Aerobic Exercise. Shea Teresi. For

Branched Chain Amino Acid, Leucine: The Effects of Leucine on Skeletal Tissue in Relation to Aerobic Exercise. Shea Teresi. For Branched Chain Amino Acid, Leucine: The Effects of Leucine on Skeletal Tissue in Relation to Aerobic Exercise By Shea Teresi For Dr. William R. Proulx, RD Associate Professor of Nutrition & Dietetics In

More information