FIG 4.
Rate of substrate hydrolysis by HOV Pro, HOV Pro R112A, and NV Pro at pH 8.0 and pH 6.5. Substrate hydrolysis was measured by FRET by mixing the protease at increasing concentrations with the fluorogenic peptide substrate and measuring the increase in fluorescence intensity as the substrate was cleaved. Based on these measurements, the Km and kcat values were calculated using GraphPad Prism 7. All experiments were carried out in triplicate. The standard errors of Km and kcat were generated by GraphPad software, and the standard errors of kcat/Km were calculated according to the Fenner formula (58).