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. Author manuscript; available in PMC: 2014 Mar 6.
Published in final edited form as: J Am Chem Soc. 2013 Feb 19;135(9):3485–3493. doi: 10.1021/ja310180c

Fig. 4.

Fig. 4

Determination of the forward commitment factor (Cf) by isotope trapping for the pyrophosphorolysis of NMN catalyzed by HsNAMPT. (a) Determination of the NMN dissociation constant at 37 °C and pH 6.30 (MES). The dissociation constant between NMN and NAMPT (Kd = 34 ± 2 μM) allows determination of the E•NMN complex concentration (by mixing 50 μM of enzyme with 175 μM of radioactive NMN, the E•NMN complex concentration becomes equal to 39.95 μM; Supporting Information Materials and Methods). (b) The NMN substrate inhibition. The isotope trapping method, used to determine Cf, is performed at high concentration of NMN; yet, this method is irrelevant in the presence of a substrate inhibition (red zone). Here, a 1 mM NMN concentration is still suitable (Supporting Information Materials and Methods). (c) Isotope trapping experiment. The synthesis of PRPP is monitored over time. After extrapolation to the origin ([PRPP] = 1.156 ± 0.008 μM), Eq. 2–4 allow determination of the Cf value (Y = 1.156 / 39.95 = 0.0289 and Cf = 0.0298 ± 0.0003; see Methods). Plots were made with KaleidaGraph 3.6 (Synergy Software).