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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Biochem J. 2013 Apr 1;451(1):69–79. doi: 10.1042/BJ20121541

Figure 6. FosB reaction mechanism analysis.

Figure 6

(A) The FosB reaction with BSH and fosfomycin follows an ordered mechanism as predicted by the double-eciprocal plots of the substrate saturation assay. Fosfomycin concentrations: ○, 1 mM; ●, 2 mM; □, 3 mM; ■ 5 mM; Δ, 8 mM; ▲ 12.5 mM; ∇ 25 mM. Inhibition studies with BOH (structure pictured) show that the SaFosB reaction is compulsory ordered with fosfomycin binding first, and that (B) BOH is competitive with respect to BSH and (C) uncompetitive with respect to fosfomycin. BOH concentrations: ○, 0 mM; ●, 1 mM; □, 3 mM; ■ 8 mM. Results are means ± S.E.M. of three replicates. Assay conditions for (A): 50 mM Hepes (pH 7.0), BSH (0.1, 0.2, 0.3, 0.5, 0.7, 1 and 2 mM), fosfomycin (1, 2, 3, 5, 8, 12.5 and 25 mM), 5 mM MnCl2 and 50 nM SaFosB. Assay conditions for (B): 150 mM Hepes (pH 7.0), 5 mM MnCl2, 15 mM fosfomycin, 50 nM FosB, 0.125–6 mM BSH and 0, 1, 3 or 8 mM BOH. Assay conditions for (C): 150 mM Hepes (pH 7.0), 5 mM MnCl2, 2.5 mM BSH, 50 nM FosB, 2–75 mM fosfomycin and 0, 1, 3 or 8 mM BOH.