Figure 4.

Understanding %²H incorporation of enzymes with opposing facioselectivity. (A) When ADH enzymes with opposite facioselectivity are supplied with [4S-²H]-NADH, ^xH^– is removed from either the S or R face of the cofactor and transferred to the substrate (acetophenone). This leads to generation of either a ²H-labeled product or an unlabeled product (with the ²H label remaining on the oxidized cofactor). (B) If access to the [S-²H]-labeled alcohol is required, the cofactor can be recycled in situ. The first cofactor turnover leads to unlabeled product (i), and further cofactor turnovers proceed via [4-²H₂]-NADH and lead to labeled product (ii). (C) The Bio system was used to supply labeled cofactor to (S)-ADH with a range of cofactor/substrate ratios, and the resulting solutions were analyzed by ¹H NMR spectroscopy (see Figure S11 in the Supporting Information). Increasing the cofactor turnover number was found to increase ²H incorporation into the phenylethanol product, tending toward 100%. Reactions were conducted on a 0.5 mL scale in ²H₅-Tris-²HCl (100 mM, p²H 8.0) with 400 μg of the Bio system as the catalyst and an excess (500 μg) of (S)-ADH being shaken under a steady stream of H₂ gas at 20 °C. In all of the reactions the initial loading of acetophenone was kept constant at 10 mM, with 2 vol % ²H₆-DMSO as a cosolvent. The starting concentration of NAD⁺ was then varied from 0.1 to 25 mM to give varying turnover numbers, with all reactions giving conversions >70% after 16 h.