Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay

We investigate how to characterize the kinetic parameters of an aminotransaminase using a non-standard coupled (or auxiliary) enzyme assay, where the peculiarity arises for two reasons. First, one of the products of the auxiliary enzyme is a substrate for the primary enzyme and, second, we explicitly account for the reversibility of the auxiliary enzyme reaction. Using singular perturbation theory, we characterize the two distinguished asymptotic limits in terms of the strength of the reverse reaction, which allows us to determine how to deduce the kinetic parameters of the primary enzyme for a characterized auxiliary enzyme. This establishes a parameter-estimation algorithm that is applicable more generally to similar reaction networks. We demonstrate the applicability of our theory by performing enzyme assays to characterize a novel putative aminotransaminase enzyme, CnAptA (UniProtKB Q0KEZ8) from Cupriavidus necator H16, for two different omega-amino acid substrates.