Fig. 1: Two enzymes, a reference enzyme (Eref, i.e., SmAA10A or HRP) and an LPMO of interest compete for the H2O2 co-substrate.

The rate of the reference reaction (vref) is a function of the concentration of the LPMO of interest. Provided with the kcat/Km (for H2O2) value for reference reaction one can calculate the kcat/Km (for H2O2) value for the LPMO reaction (oxidation of substrate, S). Note that, in the case of HRP, the reference substrate (Sref) is oxidized but the oxygen atom is not incorporated into Sref.