Enzymatic activity assay

The enzymatic activities of the WT and variant UCH-L1 were measured via hydrolysis of the fluorogenic substrate ubiquitin 7-amino-4-methylcoumarin (ubiquitin-AMC). Stock solutions of enzyme and substrate were manually mixed and diluted such that the final concentrations of ubiquitin-AMC and UCH-L1 were 500 and 25 nM, respectively. The reaction was monitored in a fluorimeter using an excitation wavelength of 380 nm and emission wavelength of 460 nm. Kinetic data obtained under steady-state conditions were fitted to a single-exponential equation to obtain a reaction rate constant.