Table 2.

Pre-steady-state kinetic analysis for USP2-catalyzed hydrolysis of Ub-AMC

rate	best fit	lower limit	upper limit
k 1 a	0.33 μM−1s−1	n/a	n/a
k −1 a	1.82 s−1	n/a	n/a
k 2	4.40 ± 0.04 s−1	4.02 s−1	5.14 s−1
k −2	1.74 ± 0.08 s−1	0.89 s−1	6.65 s−1
k 3	0.51 ± 0.01 s−1	0.33 s−1	1.24 s−1
k 4 a	2.64 s−1	n/a	n/a
k −4 a	0.40 μM−1s−1	n/a	n/a
F(Ub-AMC) a	0.61	n/a	n/a
F(USP2•Ub-AMC)	2.58 ± 0.03	2.32	2.71
F(AMC)	3.64 ± 0.01	3.63	3.65
χ2/ndf	38	n/a	n/a

^aIndicates the value was fixed during global fitting. The values for k₁, k₋₁, k₄, and k₋₄, were derived as previously explained for the USP2/Ub-AMC and the USP2/ubiquitin stopped-flow binding data (Figure 3 and Table 1). Fluorescence output expression factors (F) for Ub-AMC, USP2•Ub-AMC, and AMC are also listed within the table. The fluorescence factor for Ub-AMC was determined based on the observed fluorescence signal and the known concentration. The factor for Ub-AMC was fixed during the global fitting. The remaining rates and fluorescence factors were solved by fitting the fluorescence data in Figure 7A to reaction Scheme 1 using the KinTek Explorer software. The upper and lower limits were determined using the FitSpace Editor function of the KinTek Explorer program and the overall goodness of the fit was judged by Chi²/degrees of freedom (χ²/ndf).