A continuous kinetic assay for adenylation enzyme activity and inhibition

作者：Wilson Daniel J; Aldrich Courtney C^*

来源：Analytical Biochemistry, 2010, 404(1): 56-63.

DOI：10.1016/j.ab.2010.04.033

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

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.

• 出版日期2010-9-1