To gain better insight into the reaction mechanism catalysed by AcmB, we decided to determine the kinetic isotope effect at pH 6.5 and 8.5 (Table ). We applied two methods: i) a direct method that compared enzyme activities with the unlabelled or labelled substrates measured independently under steady-state and substrate saturation conditions and ii) a competitive method where the equimolar mixture of the unlabelled and labelled substrate was converted by the enzyme while we analysed the composition of the product with LC-MS (Figure ). The former method yields a ratio of kcat in steady-state which is dependent on both substrate oxidation and enzyme reoxidation, while the latter method also takes into account the differences in Km of the substrate isotopologues yielding 𝐷 ( 𝑉 𝐾 ). We observed a very small KIE at pH 6.5 regardless of the substitution position (1.05 and 1.17 respectively for substrate deuterated at C1 and C2, Fig. ). At pH 8.5, the observed KIE for C1 substituted substrate turned out to be slightly higher (1.33) compared to the C2 substituted substrate (1.06). The competitive experiments yielded D (V/K) values slightly higher but still in the range of only 1.2-1.5. The experimental results on KIE were confronted with the modelling and theoretical prediction of intrinsic KIE associated with particular molecular steps, as well as the overall KIE associated with the RHR process estimated with the use of free energy barriers and the Eyring equation (Tables ).