. Author manuscript; available in PMC: 2021 Apr 14.

Published in final edited form as: J Am Chem Soc. 2021 Mar 30;143(13):5038–5043. doi: 10.1021/jacs.1c00076

Table 1.

Deuterium incorporation into 5′-dAdo (9) during the incubation of substrate analogues with AprD4/AprD3.

Entry	Substrate	obs. A₂₅₃/A₂₅₂^a	% Fraction of labeled 5′-dAdo^b
1	1	0.105 ± 0.001	NA
2	14	0.101 ± 0.001	NA
3	15	0.113 ± 0.000	0.8 ± 0.1
4	15 (5 mM)	0.179 ± 0.001	6.9 ± 0.1
5	16	0.276 ± 0.003	14.6 ± 0.3
6	16 (5 mM)	0.474 ± 0.005	27.0 ± 0.4

The MS signal for protonated 5′-dAdo at 253.1 m/z (m + 1) was integrated and divided by that at 252.1 m/z (m) to obtain the observed signal intensity ratio A₂₅₃/A₂₅₂. This ratio largely reflects natural abundance ¹³C in the absence of labeled substrate (entry 1). Therefore, the ratio R of C5′-deuterated versus unlabeled 5′-dAdo observed in the presence of labeled substrate can be obtained by subtracting from the observed A₂₅₃/A₂₅₂ ratio that of entry 1.⁴⁵

The fraction of labeled 5′-dAdo is then given by R/(R+1) and is listed as a percentage (NA indicates no significant labeling). All ratios were measured from three separate assays each.