Although enzymatic activity and binding affinity are not expected to be directly correlated, the trends for specificity would likely be similar. The NMR assay is a highly quantitative method of observing product concentration and/or substrate reduction directly by NMR signal volumes throughout the reaction27. Thus A3A’s deamination activity was determined in the context of variations of the signature sequence (T/C)TC(A/G) using a 1H NMR based A3 deaminase activity assay. High (TTCA and TTCG), medium (ATCA, ATCG, GTCA, GTCG, TTCT) and low (ATCT and GTCT) affinity sequences were tested (Table 2) to determine the correlation between binding and activity. Overall, activity by NMR has the same trend as affinity from the binding assay (Fig. 4). This indicates that in general those substrates sequences with varying binding affinity (high, medium and weak) are also processed in a similar order.Table 2A3A enzyme activity for DNA sequences.DNA sequenceActivity (min−1) 40 °CPoly A-ATCA27 ± 1Poly A-ATCG28 ± 1Poly A-ATCT30 ± 2Poly A-GTCA38 ± 2Poly A-GTCG31 ± 2Poly A-GTCT22 ± 2Poly A-TTCA52 ± 2Poly A-TTCG36 ± 1Poly A-TTCT37 ± 2Figure 4Binding affinity versus enzyme activity. The enzyme activity of active A3A measured by NMR-based deamination assay versus the free energy of binding calculated [ΔG = −RTln (Kd)] from the binding affinity for nine 12-mers. These nine represent 2 high binding (green), 5 medium binding (orange) and 2 weak binding (red) sequences.