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. 2009 Dec 25;66(Pt 1):2–7. doi: 10.1107/S1744309109044741

Figure 2.

Figure 2

Proposed catalytic reaction of paAzoRs. (a) Azoreductase reduces methyl red to N,N′-dimethyl-p-phenylenediamine and 2-aminobenzoic acid with the consumption of 2 mol NAD(P)H. FMN serves as the electron mediator between NAD(P)H and methyl red in a proposed double ping-pong bi-bi biomechanism. (b) Lineweaver–Burk plots showing the ping-pong bi-bi mechanism in the azoreduction of methyl red by paAzoR1 with NADPH as the electron donor. The plots show the effect of varying the methyl red concentration; the concentrations tested were 5, 10, 15 and 20 µM in 20 mM Tris–HCl buffer containing 0.3 M NaCl and 5 µg paAzoR1. The reactions were initiated with NADPH at concentrations of 0.2 mM (solid squares), 0.3 mM (open squares), 0.4 mM (solid triangles) and 0.5 mM (open triangles) in a total volume of 200 µl. (c) Lineweaver–Burk plots showing the ping-pong bi-bi mechanism in the azoreduction of methyl red by paAzoR1 with NADPH as the electron donor. The plots show the effect of varying the concentration of NADPH. The NADPH concentrations tested were 0.2, 0.3, 0.4, 0.5 and 2 mM in 20 mM Tris–HCl buffer, 0.3 M NaCl. The reactions were initiated by adding NADPH to a reaction mixture containing 5 µg paAzoR1 and methyl red at 5 µM (solid squares), 15 µM (open squares), 20 µM (solid triangles) or 25 µM (open triangles) in a total volume of 200 µl. In each case the rate is the initial rate of reduction of methyl red.