Figure 3.

Time courses of enzymatic oxidations of UDP-Glc, and determination of the associated change of relative H-5 signal intensity in UDP-GlcUA product. Time courses are shown in panels A (wild-type) and C (E161Q), the symbols used are square (UDP-Glc), triangle (UDP-GlcUA), and diamond (NADH). The low conversion can be explained by a large solvent isotope effect on k_cat (see text). Two equivalents of NADH are formed per equivalent UDP-Glc utilized, as expected. Proton signal intensities in UDP-GlcUA are shown in panels B and D (square: H-1; triangle H-4; diamond: H-5) for reactions with wild-type enzyme and E161Q mutant, respectively. In case of deuterium incorporation at C-5, one would see a decrease of the respective proton signal compared to H-1 and H-4.