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.

我们研究如何使用非标准耦合（或辅助）酶测定来表征转氨酶的动力学参数，其中出现的特殊性有两个原因。首先，辅助酶的产物之一是主要酶的底物，其次，我们明确说明了辅助酶反应的可逆性。使用奇异扰动理论，我们根据逆反应的强度来表征两个不同的渐近极限，这使我们能够确定如何推导表征辅助酶的主要酶的动力学参数。这建立了一个更普遍适用于类似反应网络的参数估计算法。