Techniques for Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl‐CoA:Amino Acid N‐Acyltransferase: The Amino Acid Conjugation Pathway

Michael Kelley1, Donald A. Vessey1

1 Veterans Administration Medical Center, San Francisco, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.11
DOI:  10.1002/0471140856.tx0411s14
Online Posting Date:  February, 2003
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Abstract

A wide variety of xenobiotic carboxylic acids are metabolized to their amino acid conjugates via a pathway that exists primarily in liver and kidney. This conjugation occurs in a two‐step pathway catalyzed by two distince types of enzymes, ligases and transferases. Measurements of acyl‐CoA ligase activity include monitoring the rate of appearance of AMP or PPi, or the CoA adduct. N‐acyltransferases catalyze formation of an amino acid conjugate from the CoA‐activated intermediate, releasing CoA. This reaction is monitored by following the release of free CoA or the disappearance of the acyl‐CoA adduct.

     
 
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Table of Contents

  • Basic Protocol 1: Direct Measurement of Carboxylic Acid:CoA Ligase Activity using a Radiolabeled Carboxylic Acid
  • Alternate Protocol 1: Measurement of Radiolabeled ATP Cleavage by Carboxylic Acid:CoA Ligase
  • Alternate Protocol 2: Spectrophotometric Assay of Carboxylic Acid:CoA Ligase Activity using DTA
  • Alternate Protocol 3: Indirect Assay of Carboxylic Acid:CoA Ligase using AMP Formation Coupled to NADH Oxidation
  • Alternate Protocol 4: Monitoring Carboxylic Acid:CoA Ligase Activity by HPLC Determination of Acyl‐CoA Product Formation
  • Basic Protocol 2: Monitoring CoA Release using a DTNB‐coupled Assay to Determine N‐Acylasetransferase Activity
  • Alternate Protocol 5: Direct Assay of N‐Acyltransferase Activity by Monitoring the Disappearance of Thioester Bond Absorbance
  • Alternate Protocol 6: Radioactive Assays for N‐Acyltransferases
  • Alternate Protocol 7: HPLC Assays for N‐Acyltransferases
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Direct Measurement of Carboxylic Acid:CoA Ligase Activity using a Radiolabeled Carboxylic Acid

  Materials
  • Kill/acidification mix (see recipe) in dispenser set to deliver 0.4 ml
  • Water‐saturated butanol (see recipe) in dispenser set to deliver 1 ml
  • 1 M Tris⋅Cl, pH 8.0 at 30°C ( appendix 2A)
  • 1 M KCl
  • 100 mM MgCl 2
  • 5 mM coenzyme A (see recipe)
  • Radiolabeled carboxylic acid substrate (e.g., 100 µM 80 cpm/mol [14C]benzoate; see recipe)
  • 1 mM ATP (see recipe)
  • Carboxylic acid:CoA ligase preparation (see )
  • Scintillation fluid
  • Positive‐displacement pipet (e.g., Drummond)
  • 30°C water bath
  • Vacuum pipettor
  • Scintillation vials
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A). Also note that short‐chain fatty acids are volatile and should be maintained in sealed tubes to avoid contamination of storage areas.

Alternate Protocol 1: Measurement of Radiolabeled ATP Cleavage by Carboxylic Acid:CoA Ligase

  • ATP‐assay kill mix (50 mM Na 2EDTA/5 mM K 2HPO 4) in a dispenser set to deliver 1.9 ml
  • Unlabeled carboxylic acid substrate (e.g., 1 mM hydrocinnamic acid)
  • [γ‐33 P]ATP of known concentration and specific activity (e.g., 10 mM, 6.8 cpm/pmol; also see recipe for radiolabeled carboxylic acid substrates for specific activity determination)
  • Phosphoric and sulfuric acid‐washed activated charcoal (e.g., Sigma)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A).

Alternate Protocol 2: Spectrophotometric Assay of Carboxylic Acid:CoA Ligase Activity using DTA

  • 5 mM DTA (see recipe)
  • Recording spectrophotometer, thermostatically‐controlled at 30°C

Alternate Protocol 3: Indirect Assay of Carboxylic Acid:CoA Ligase using AMP Formation Coupled to NADH Oxidation

  Materials
  • 1 mM DTNB (see recipe)
  • 1 M KCl
  • 1 mM benzoyl‐CoA
  • N‐acyltransferase preparation (see )
  • Acceptor amino acid (i.e., glycine or glutamine)
  • Recording spectrophotometer, thermostatically‐controlled at 30°C

Alternate Protocol 4: Monitoring Carboxylic Acid:CoA Ligase Activity by HPLC Determination of Acyl‐CoA Product Formation

  • 0.5 M Tris⋅Cl, pH 8.0 at 30°C ( appendix 2A)
  • 1‐ml quartz cuvettes
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Figures

Videos

Literature Cited

Literature Cited
   Bronfman, M., Amigo, L., and Morales, M.N. 1986. Activation hypolipidaemic drugs to acylcoenzyme A thioesters. Biochem. J. 239:781‐784.
   Brugger, R., Alia, B.G., Reichel, C., Waibel, R., Menzel, S., Brune, K., and Geisslinger, G. 1996. Isolation and characterization of rat liver microsomal R‐ibuprofenoyl‐CoA synthethase. Biochem. Pharmacol. 52:1007‐1013.
   Dawson, A., Elliott, D.C., Elliott, W.H., and Jones, K.M. 1986. Data for Biochemical Research, Third Edition. Clarendon Press, Oxford.
   Garland, P.B., Yates, D.W., and Haddock, B.A. 1970. Spectrophotometric studies of acyl‐coenzyme A synthetases of rat liver mitochondria. Biochem. J. 119:553‐564.
   Gregus, Z., Fekete, T., Halaszi, E., and Klaassen, C.D. 1996. Lipoic acid impairs glycine conjugation of benzoic acid and renal excretion of benzoylglycine. Drug Metab. Disp. 24:682‐688
   James, M.R. and Bend, J.R. 1978. A radiochemical assay for glycine N‐acyltransferase activity. Biochem. J. 172:285‐291.
   Kasuya, F., Igarashi, K., and Fukui, M. 1990. Glycine conjugation of the substituted benzoic acids in vitro: Structure‐metabolism relationship study. J. Pharmacobio. Dyn. 13:432‐440.
   Kasuya, F., Igarashi, K., Fukui, M., and Nokihara, K. 1996. Purification and characterization of a medium chain acyl‐coenzyme A synthetase. Drug Metab. Dispos. 24:879‐883.
   Kelley, M. and Vessey, D.A. 1990. The effects of ions on the conjugation of xenobiotics by the aralkyl‐CoA and arylacetyl‐CoA N‐acyltransferases from bovine liver mitochondria. J. Biochem. Tox. 5:125‐135.
   Knights, K.M. and Roberts, B.J. 1994. Xenobiotic acyl‐CoA formation: Evidence of kinetically distinct hepatic microsomal long‐chain fatty acid and nafenopin‐CoA ligases. Chemico‐Biol. Interact. 90:215‐223.
   Rodriguez‐Aparicio, L.B., Reglero, A., Martinex‐Blanco, H., and Luengo, J.M. 1991. Fluorimetric determination of phenylacetylCoA ligase from Pseudomonas putida: A very sensitive assay for a newly described enzyme. Biochim. Biophys Acta 1073:431‐433.
   Vessey, D.A. 1997. Enzymes involved in the formation of amide bonds. In Comprehensive Toxicology Vol. 3, Biotransformation (Guengerich, F.P., ed.), p. 455‐475. Elsevier Science, Oxford.
   Vessey, D.A., Crissey, M.H., and Zakim, D. 1977. Kinetic studies on the enzymes conjugating bile acids with taurine and glycine in bovine liver. Biochem. J. 163:181‐183.
   Webster, L.T. Jr., Siddiqui, U.A., Lucas, S.V., Strong, J.M., and Mieyal, J.J. 1976. Identification of separate acyl‐CoA:glycine and acyl‐CoA:glutamine N‐acyltransferase activities in mitochondrial fractions from liver of rhesus monkey and man. J. Biol. Chem. 251:3352‐3358.
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