Kinetic Characterization of Deaminase Enzymes

Deaminase activity was measured for EcGuaD and Gud1 using a previously described assay that couples ammonia release to nicotinamide adenine dinucleotide (NADH) oxidation.^{19 (link), 20 (link)} All kinetic assays were performed at 37°C. Substrate screening was carried out at two concentrations, 200 μM and 1 mM. The coupled assay was typically run with 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.2 mM NADH, 1 mM α-ketoglutarate, and 4 units of glutamate dehydrogenase (Sigma). The reaction was initiated by the addition of enzyme and the resulting decrease in absorbance at 340 nm was monitored using a Synergy Neo2 microplate reader (Biotek). The enzyme concentration ranged between 0.4 μM and 3.7 μM. The kinetic parameters k_cat, k_cat/K_m and K_m were determined by plotting the initial rates (less than 10% reaction completion) as a function of substrate concentration and fitting the data using the Michaelis−Menten equation. All kinetic data were analyzed using Graphpad Prism 4 or KaleidaGraph (Synergy Software). Enzyme concentrations were determined using the Bradford assay.