A continuous kinetic assay for adenylation enzyme activity and inhibition.
Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PP(i)). 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-PP(i) exchange.
Wilson
Daniel J
DJ
Academic Health Center, University of Minnesota, Minneapolis, 55455, USA.
Aldrich
Courtney C
CC
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D055162:Biocatalysis; D011756:Diphosphates / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D006877:Hydroxamic Acids / Q000378:metabolism; D007700:Kinetics; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*
D001426:Bacterial Proteins; D011756:Diphosphates; D006877:Hydroxamic Acids; D000217:Acyltransferases; D011683:Purine-Nucleoside Phosphorylase; D011755:Pyrophosphatases
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