Enzymatic Kinetics and Michaelis-Menten Analysis

Enzyme kinetics and the Michaelis–Menten parameters were determined as described previously^{32 (link)}. Briefly, recombinant NAs at a fixed concentration of 10 µg/ml were incubated with 1.5 fold dilutions of the fluorogenic MUNANA substrate in MES (2-ethanesulfonic acid2-ethanesulfonic acid) buffer with suitable blank controls for background fluorescence. The plates were incubated at 37 °C and readings for relative fluorescence units (RFUs) were recorded at every 90 s for 40 min using a Gen5 v3.0 Software and a Synergy H1 Microplate Reader (BioTek). The RFU readings were captured at excitation and emission wavelengths of 360 and 448 nM. Velocity of the reaction was determined by plotting the RFU readings against time, and the Michaelis–Menten parameters V_max and K_m were determined through non-linear regression fits of the velocity and MUNANA concentrations in Graphpad Prism 7.