A continuous kinetic assay for adenylation enzyme activity and inhibition

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Abstract

Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PPi). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hydroxylamine as a surrogate acceptor molecule, leading to the formation of a hydroxamate. The released pyrophosphate from the first half-reaction is measured using the pyrophosphatase-purine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG). The coupled hydroxamate-MesG assay is especially useful for characterizing the activity and inhibition of adenylation enzymes that acylate a protein substrate and/or fail to undergo rapid ATP-PPi exchange.

Original languageEnglish (US)
Pages (from-to)56-63
Number of pages8
JournalAnalytical Biochemistry
Volume404
Issue number1
DOIs
StatePublished - Sep 2010

Keywords

  • Adenylate-forming
  • Adenylation
  • Enzyme assay
  • Hydroxamate
  • MesG

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