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. Author manuscript; available in PMC: 2020 Feb 12.
Published in final edited form as: Biochemistry. 2018 Dec 27;58(6):665–678. doi: 10.1021/acs.biochem.8b00716

Figure 2: Adenylation activity of PchF variants.

Figure 2:

PchF catalyzes the adenylation of l-cysteine by ATP forming an aminoacyl-adenylate bond, releasing pyrophosphate. In the adenylation assay (Figure S3), pyrophosphate is converted to two inorganic phosphates by inorganic pyrophosphatase. 7-methylguanosine is then phosphorylated by purine nucleoside phosphorylase to generate 7-methylguanine, which absorbs at 360 nm. A) PchF variants do not turnover with l-cysteine and ATP (A), but do with the addition of hydroxylamine as a nucleophilic surrogate (B). Michaelis-Menten steady-state kinetics (C) were performed to compare the l-cysteine adenylation activity of PchF-FL, PchF-AMT, and the methyltransferase null mutant of G667I-PchF-AMT.