Figure 5. Demonstration of the adherence to the Michaelis–Menten equation of (a) catalysis of NAD⁺ and (b) ligation of the AE-labelled DNA substrate by S. aureus DNA ligase.

(a) Assays were carried out in Hepes (pH 7.4) assay buffer containing 10 nM AE-labelled DNA substrate and 50 pM enzyme, with variation in NAD⁺ concentration between 2 and 45 μM. The points are experimental and the continuous lines correspond to v_i=V_max[S]₀/(K_m+[S]₀) with K_m=2.75±0.1 μM and V_max=(6.20±0.05)×10⁻¹³ M/s. (b) Assays were carried out in Hepes (pH 7.4) assay buffer containing 25 μM NAD⁺, 50 pM enzyme, with variation in the concentration of AE-labelled DNA between 1 and 10 nM. The points are experimental and the continuous lines correspond to v_i=V_max[S]₀/(K_m+[S]₀) with K_m=2.5±0.2 nM and V_max=(6.15±0.05)×10⁻¹³ M/s.