Steady-state ATPase activity of β-cardiac myosin constructs. (a–d) Steady-state ATPase kinetics of WT and E525K β-cardiac myosin constructs were determined using an NADH-coupled assay across varying actin concentrations. E525K mutations in S1 and 2HEP constructs significantly increased actin-activated ATPase activity (∼3-fold) and decreased the actin concentration for half-maximal activity (K_ATPase) by ∼18-fold. In contrast, 15HEP and 25HEP constructs showed reduced ATPase activities, with no significant difference between WT and E525K. Data are presented as mean ± SD of three experiments from separate protein preparations. Data in c originally published in Rasicci et al. (2022). Comparison p-values provided in Tables S1 and S2.