Branched-Chain Amino Acids, Part B (eBook)

Robert Adron Harris, John R. Sokatch (Herausgeber)

eBook Download: PDF

2000 | 1. Auflage
550 Seiten
Elsevier Science (Verlag)
978-0-08-049679-5 (ISBN)

Infos

Volume 324 of Methods in Enzymology supplements Volume 166. It includes genetic information (cloning, gene expression) and information on human genetic diseases not available when Volume 166 was published.
General Description of the Series:
The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.

Key Features
* Preparation of substrates and assay of enzymes
* Cloning, expression, and purification of enzymes
* Detection and consequences of genetic defects
* Regulation and expression of enzymes

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Volume 324 of Methods in Enzymology supplements Volume 166. It includes genetic information (cloning, gene expression) and information on human genetic diseases not available when Volume 166 was published.General Description of the Series:The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences. - Preparation of substrates and assay of enzymes- Cloning, expression, and purification of enzymes- Detection and consequences of genetic defects- Regulation and expression of enzymes

Inhalt

Front Cover 1
Branched-Chain Amino Acids 4
Copyright Page 5
Table of Contents 6
Contributors to Volume 324 12
Preface 16
Volumes in Series 18
Section I: Preparation of Substrates, Assays of Intermediates and Enzymes, and Use of Enzyme Inhibitors 37
Chapter 1. Synthesis and Gas Chromatography/Mass Spectrometry Analysis of Stereoisomers of 2-Hydroxy- 3-methylpentanoic Acid 39
Chapter 2. Analysis of Intracellular Metabolites as Tool for Studying Branched-Chain Amino Acid Biosynthesis and Its Inhibition in Bacteria 46
Chapter 3. Determination of Branched-Chain L-Amino-Acid Aminotransferase Activity 59
Chapter 4. Analysis of (S)- and (R)-3-Methyl-2-oxopentanoate Enantiomorphs in Body Fluids 69
Chapter 5. Spectrophotometric Assay for Measuring Branched-Chain Amino Acids 76
Chapter 6. Determination of Branched-Chain a-Keto Acid Dehydrogenase Activity State and Branched-Chain a-Keto Acid Dehydrogenase Kinase Activity and Protein in Mammalian Tissues 84
Chapter 7. Simultaneous Quantification of Plasma Levels of a-Ketoisocaproate and Leucine by Gas Chromatography–Mass Spectrometry 98
Chapter 8. Synthesis of Methacrylyl-CoA and (R)- and (S)- Hydroxyisobutyryl-CoA 109
Chapter 9. Pathways of Leucine and Valine Catabolism in Yeast 116
Section II: Cloning, Expression, and Purification of Enzymes of Branched-Chain Amino Acid Metabolism 129
Chapter 10. Isolation of Subunits of Acetohydroxy Acid Synthase Isozyme III and Reconstitution of Holoenzyme 131
Chapter 11. Branched-Chain Amino-Acid Aminotransferase of Escherichia coli 139
Chapter 12. Purification of Sodium-Coupled Branched-Chain Amino Acid Carrier of Pseudomonas aeruginosa 150
Chapter 13. Reconstitution of Pseudomonas aeruginosa High-Affinity Branched-Chain Amino Acid Transport System 158
Chapter 14. Purification of Pseudomonas putida Branched-Chain Keto Acid Dehydrogenase E1 Component 165
Chapter 15. Pseudomonas mevalonii 3-Hydroxy-3-methylglutaryl-CoA Lyase 175
Chapter 16. Human 3-Hydroxy-3-methylglutaryl-CoA Lyase 186
Chapter 17. Branched-Chain a-Keto Acid Dehydrogenase Kinase 198
Chapter 18. Expression of E1 Component of Human Branched- Chain a-Keto Acid Dehydrogenase Complex in Escherichia coli by Cotransformation with Chaperonins GroEL and GroES 215
Chapter 19. Production of Recombinant Mammalian Holo-E2 and E3 and Reconstitution of Functional Branched-Chain a-Keto Acid Dehydrogenase Complex with Recombinant E1 228
Chapter 20. Production of Recombinant E1 Component of Branched-Chain a-Keto Acid Dehydrogenase Complex 236
Chapter 21. Mammalian Methylmalonate-Semialdehyde Dehydrogenase 243
Chapter 22. Mammalian 3-Hydroxyisobutyrate Dehydrogenase 254
Chapter 23. 3-Hydroxyisobutyryl-CoA Hydrolase 265
Chapter 24. Mammalian Branched-Chain Acyl-CoA Dehydrogenases: Molecular Cloning and Characterization of Recombinant Enzymes 277
Chapter 25. 3-Hydroxy-3-methylglutaryl-CoA Reductase 295
Chapter 26. Characterization of 3-Methylcrotonyl-CoA Carboxylase from Plants 316
Chapter 27. Purification of D-Hydroxyisovalerate Dehydrogenase from Fusarium sambucinum 329
Chapter 28. Purification and Characterization of Recombinant 3-Isopropylmalate Dehydrogenases from Thermus thermophilus and Other Microorganisms 337
Chapter 29. Wild-Type and Hexahistidine-Tagged Derivatives of Leucine-Responsive Regulatory Protein from Escherichia coli 358
Chapter 30. Purification of Branched-Chain Keto Acid Dehydrogenase Regulator from Pseudomonas putida 365
Chapter 31. Mitochondrial Import of Mammalian Branched- Chain a-Keto Acid Dehydrogenase Complex Subunits 372
Chapter 32. Cloning, Expression, and Purification of Mammalian 4-Hydroxyphenylpyruvate Dioxygenase/a-Ketoisocaproate Dioxygenase 378
Chapter 33. Mammalian Branched-Chain Aminotransferases 391
Chapter 34. Branched-Chain-Amino-Acid Transaminases of Yeast Saccharomyces cerevisiae 401
Chapter 35. Purification, Properties, and Sequencing of Aminoisobutyrate Aminotransferases from Rat Liver 412
Chapter 36. Branched-Chain Keto Acid Dehydrogenase of Yeast 425
Chapter 37. B-Alanine Synthase, an Enzyme Involved in Catabolism of Uracil and Thymine 435
Section III: Detection and Consequences of Genetic Defects in Genes Encoding Enzymes of Branched-Chain Amino Acid Metabolism 447
Chapter 38. Diagnosis and Mutational Analysis of Maple Syrup Urine Disease Using Cell Cultures 449
Chapter 39. Detection of Gene Defects in Branched-Chain Amino Acid Metabolism by Tandem Mass Spectrometry of Carnitine Esters Produced by Cultured Fibroblasts 460
Chapter 40. Molecular and Enzymatic Methods for Detection of Genetic Defects in Distal Pathways of Branched- Chain Amino Acid Metabolism 468
Chapter 41. Genetic Defects in E3 Component of a-Keto Acid Dehydrogenase Complexes 489
Chapter 42. Targeting E3 Component of a-Keto Acid Dehydrogenase Complexes 501
Section IV. Regulation and Expression of Enzymes of Branched-Chain Amino Acid Metabolism 513
Chapter 43. Regulation of Expression of Branched-Chain a-Keto Acid Dehydrogenase Subunits in Permanent Cell Lines 515
Chapter 44. Expression of Murine Branched-Chain a-Keto Acid Dehydrogenase Kinase 527
Chapter 45. Regulation of Branched-Chain a-Keto Acid Dehydrogenase Kinase Gene Expression by Glucocorticoids in Hepatoma Cells and Rat Liver 534
Author Index 549
Subject Index 571

Textauszug

[1]

Synthesis and Gas Chromatography/Mass Spectrometry Analysis of Stereoisomers of 2-Hydroxy-3-methylpentanoic Acid

Orval A. Mamer

Introduction

2-Hydroxy-3-methylpentanoic acid (HMPA) is a familiar metabolite of isoleucine commonly found in normal human urine, plasma, and other fluids. In disorders affecting branched-chain amino acid metabolism, such as branched-chain ketoaciduria, HMPA is frequently elevated in these fluids, sometimes by two orders of magnitude or more. HMPA is formed by the reduction of 2-keto-(3S)-methylpentanoic acid [(3S)-KMPA], which is the transamination product of L-isoleucine in the degradative pathway of that amino acid.1,2 The chiral center of KMPA is spontaneously racemized in solution at high pH through keto–enol tautomerism1 (Fig. 1).

Fig. 1 Racemization of KMPA by enolization at high pH in aqueous solution.

Having two chiral centers, HMPA exists in four isomeric forms (Fig. 2): (2R)-hydroxy-(3R)-methylpentanoic acid [(2R,3R)-HMPA], (2S)-hydroxy- (3S)-methylpentanoic acid [(2S,3S)-HMPA], (2R)-hydroxy-(3S)-methylpentanoic acid [(2R,3S)-HMPA], and (2S)-hydroxy-(3R)-methylpentanoic acid [(2S,3R)-HMPA]. Each of these may be synthesized in pure form by conventional diazotization of the isomer of 2-amino-3-methylpentanoic acid having the corresponding steric configuration and trivial or common names of D-isoleucine, L-isoleucine, D-alloisoleucine, and L-alloisoleucine, respectively. All four of these amino acids are commercially available in relatively pure form.

Fig. 2 The four stereoisomers of HMPA.

Synthesis

General

All solutions are aqueous unless stated otherwise, reactions are carried out in a fume hood to reduce exposure to hazardous vapors, organic chemicals are from Sigma-Aldrich Canada (Oakville, ON, Canada), and inorganic materials are purchased from local suppliers and are used as received.

Gas Chromatography/Mass Spectrometry Analysis

Analyses are run on a Hewlett-Packard (Palo Alto, CA) model 5988A gas chromatograph/mass spectrometer (GC/MS) equipped with a 5890A gas chromatograph fitted with a 30 m × 0.25 mm (i.d.) fused silica column having a 0.25-μm dimethylsilicone coating. The helium flow rate is set at 1 ml/min. The injector port is set at 250° and configured for splitless injection of 1-μ1 aliquots of the trimethylsilyl (TMS) derivative mixtures. After a 1-min hold at 70° for septum purging, the column is programmed at 5°/min to 200°. The interface and ion source temperatures are set at 270 and 200°, respectively. Ionization is by electron impact at 70 eV, and scanning is over the range 100 to 400 Da.

Racemic 2-Hydroxy-3-methylpentanoic Acid

Fully racemic HMPA may be conveniently synthesized by the sodium borohydride reduction of KMPA racemized in solution at high pH. The sodium salt of KMPA (150 mg, 1 mmol; Sigma-Aldrich) is dissolved in 5 ml of water, a few drops of 2 N sodium hydroxide are added to raise the pH to pH > 12, and 50 mg of sodium borohydride is added with stirring until dissolution is complete. The resulting solution is held in a water bath at 60° for 2 hr, cooled in an ice–water bath, and then cautiously acidified to pH < 2 with 2 N hydrochloric acid. (Caution: Vigorous evolution of hydrogen gas occurs.) The acidified solution is saturated with sodium chloride and extracted three times with volumes of diethyl ether equal to the aqueous phase. The ether extracts are combined, treated with 200 mg of anhydrous sodium sulfate to remove water, and evaporated to an oil (approximately 100-mg yield) under a warm, dry nitrogen stream. The oil may be crystallized from an ethyl acetate–petroleum ether solvent pair if desired, although this is not necessary for further work.

A few micrograms of the product oil is converted to the TMS derivative in a mixture of 25-μl volumes of anhydrous pyridine and N,O-bis(trimethylsily)trifluoroacetamide (BSTFA) in a capped autoinjector vial, heated at 60° for 20 min, and analyzed by GC/MS as described above. The relevant portion of the resulting chromatogram is reproduced in Fig. 3A.

Fig. 3 Gas chromatograms obtained for the TMS derivatives of (A) racemic HMPA synthesized by sodium borohydride reduction of racemic KMPA, and (B) commercially available, isomerically pure (2S,3S)-HMPA.

Two unequal peaks are found for racemic HMPA made from fully racemized KMPA. Although it is not possible to separate by chromatography enantiomers in achiral stationary phases, diastereomers that are not mirror images of each other are frequently easily separable, as they have different chemical and physical properties. Sodium borohydride reduction is expected to produce less of a racemic (2S,3R)- and (2R,3S)-HMPA enantiomeric pair than of a (2S,3S)-HMPA, (2R,3R)-HMPA pair.1 This is borne out in the resulting chromatogram. The mass spectra of the two peaks are reproduced in Fig. 4A and B, and cannot be distinguished reliably.

Fig. 4 Mass spectra obtained for (A) peak 1 in Fig. 3A, (B) peak 2 in Fig. 3A, and (C) the major peak in Fig. 3B.

L-2-Hydroxy-3-methylpentanoic Acid

Sodium L-2-hydroxy-3-methylvalerate (50 mg, 0.3 mmol; Sigma) is dissolved in 2 ml of water, which is then acidified, extracted, isolated, and derivatized as described above. The resulting mixture when analyzed by GC/MS produces a single peak (Fig. 3B) having the same retention time as the second eluting peak in Fig. 3A and the mass spectrum shown in Fig. 4C. Again, the mass spectrum of this isomer is indistinguishable from those of the racemate. Because (3S)-KMPA is probably not racemized after its biosynthesis from L-isoleucine, reduction to HMPA, presumably by a lactate dehydrogenase (LDH) isozyme, will leave the S-chirality at the 3-carbon unaffected. LDH reductions of 2-keto acids produce S-chirality at the 2-carbon. This assignment of “L-HMPA” as (2S,3S)-HMPA is consistent then with its elution with the retention time of peak 2 in the racemate (Fig. 3).

2-Hydroxy-3-methylpentanoic Acid Stereoisomers

HMPA isomers may be conveniently synthesized in pure form by diazotization of the corresponding amino acid in dilute perchloric acid. Replacement of the amino group by a hydroxyl group is known to occur with retention of configuration about the 2-carbon atom, owing to anchiomeric participation of the neighboring carboxyl function1 (Fig. 5).

Fig. 5 Retention of configuration by anchiomeric participation of the carboxylate group in the diazotization of L-isoleucine.

L-Isoleucine (100 mg, 0.75 mmol is dissolved in 50 ml of cold (0°) 0.2 N perchloric acid. To this is added a cold solution (0°) of sodium nitrite (1.4 g, 20 mmol) in 20 ml of water with rapid stirring. Stirring is continued while the mixture is allowed to warm to room temperature and the evolution of gas subsides (approximately 0.5 hr). The resulting solution is raised to the boiling point for a few minutes, cooled to room temperature, and saturated with sodium chloride. Ether extracts are made and dried as described above, and the product is isolated similarly to yield 75 mg of (2S,3S)-HMPA as an oil. This product is also analyzed as the TMS derivative by GC/MS.

The three other stereoisomers, (2R,3R)-HMPA, (2R,3S)-HMPA, and (2S,3R)-HMPA, are synthesized in a similar manner from D-isoleucine, D-alloisoleucine, and L-alloisoleucine, respectively.

All four of the stereoisomers have mass spectra indistinguishable from each other.1 Relative retention times are: (2S,3S)- and (2R,3R)-HMPA coelute after (2R,3S)- and (2S,3R)-HMPA, which also coelute.

Resolution of Four Stereoisomers in Racemic Mixture

The four stereoisomers may be resolved by introducing a third chiral center (Fig. 6). For this purpose, 20 mg (0.2 mmol) of (S)-(+)-2-methylbutanoic acid is dissolved in 10 ml of toluene, 75 μl of thionyl chloride (1 mmol) is added, and the resulting solution is heated at reflux for 2 hr. While still warm, the toluene solution is reduced in volume under reduced pressure to approximately 3 ml and then added to 10 mg of racemic HMPA produced as described above and dissolved in 15 ml of toluene. A few drops of pyridine are added, and the resulting mixture is heated at reflux...

Produkt-Details

• Erscheint lt. Verlag: 6.9.2000
• Mitarbeit: Chef-Herausgeber: John N. Abelson, Melvin I. Simon
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 0-08-049679-2 / 0080496792
• ISBN-13: 978-0-08-049679-5 / 9780080496795