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. Author manuscript; available in PMC: 2020 Aug 12.
Published in final edited form as: Nature. 2020 Feb 12;578(7795):461–466. doi: 10.1038/s41586-020-2000-y

Extended Data Table 1 |.

Estimates of Km and kobs for Substrate Ubiquitylation.

Substrate E2 CRL1β-TRCP Km(10−9M) kobsS0–S1 (sec−1) kobsS1–S2 (sec−1) kobsS0–S1/ Km (M−1sec−1) Fold change
IκBα WT UBE2D NEDD8-CUL1–RBX1 408 ± 57 12.9 ± 1.7 0.24 ± 0.04 3.2 × 107 -
IκBα WT UBE2D CUL1–RBX1 4014 ±956 0.05 ± 0.002 - 1.2 × 104 2667
WT β-cat WT UBE2D NEDD8-CUL1–RBX1 214 ±30 4.8 ±0.46 0.19 ±0.02 2.2 × 107 -
WT β-cat WT UBE2D CUL1–RBX1 4638±138 0.05 ± 0.005 - 1.1 × 104 2000
medium β-cat WT UBE2D NEDD8-CUL1–RBX1 372 ± 48 11 ± 1.00 0.24 ± 0.04 3.0 × 107 -
medium β-cat WT UBE2D CUL1–RBX1 4875 ±1328 0.08 ± 0.005 - 1.6 × 104 1875
short β-cat WT UBE2D CUL1–RBX1 5207±1051 0.02 ± 0.0004 - 3.8 × 103
medium β-cat WT UBE2D NEDD8 I44A-CUL1–RBX1 1087 ±69 3.60 ±0.27 0.17 ±0.03 3.3 × 106 9.1
medium β-cat WT UBE2D UB-CUL1–RBX1 2056 ±170 1.0 ±0.03 0.15 ±0.01 4.9 × 105 61.2
medium β-cat WT UBE2D NEDD8 Q40E-CUL1–RBX1 1941 ±226 1.77 ± 0.13 0.07 ±0.01 9.1 × 105 33.0
medium β-cat WT UBE2D UBylized NEDD8-CUL1–RBX1 1651±137 2.20 ±0.15 0.17 ±0.04 1.3 × 106 23.1
medium β-cat S22R UBE2D NEDD8-CUL1–RBX1 913 ±135 1.89 ±0.14 0.17 ±0.03 2.1 × 106 14.2
medium β-cat S22R UBE2D NEDD8 I44A-CUL1–RBX1 5190 ±604 0.13 ±0.01 - 2.5 × 104 1200
medium β-cat S22R UBE2D UB-CUL1–RBX1 6492 ± 872 0.08 ± 0.005 - 1.2 × 104 2500
medium β-cat S22R UBE2D CUL1–RBX1 4007 ± 754 0.013 ±0.002 - 3.3 ×103 9091
medium β-cat S22R UBE2D NEDD8 Q40E-CUL1–RBX1 3581 ± 367 0.17 ±0.005 - 4.7 × 104 638.3
medium β-cat S22R UBE2D UBylized NEDD8-CUL1–RBX1 6105 ±1030 0.12 ±0.007 - 2.0 × 104 1500
medium β-cat H32A UBE2D NEDD8-CUL1–RBX1 347 ± 53 8.15 ± 1.04 0.73 ± 0.09 2.3 × 107 1.3
medium β-cat H32A UBE2D NEDD8 I44A-CUL1–RBX1 3431 ± 332 0.58 ± 0.04 0.12 ±0.02 1.7 × 105 176.5
medium β-cat H32A UBE2D UB-CUL1–RBX1 2999 ± 301 0.34 ±0.01 0.10 ±0.02 1.1 × 105 272.7
medium β-cat H32A UBE2D CUL1–RBX1 3305 ±1119 0.06 ± 0.003 - 1.8 × 104 1666.7
medium β-cat H32A UBE2D NEDD8 Q40E-CUL1–RBX1 2923 ± 459 0.81 ±0.06 - 2.8 × 105 107.1
medium β-cat H32A UBE2D UBylized NEDD8-CUL1–RBX1 4303 ±310 0.28 ± 0.02 - 6.5 × 104 461.5
UB-β-cat WT UBE2D NEDD8-CUL1–RBX1 148 ± 15 - 0.48 ± 0.02
UB-β-cat WT UBE2D CUL1–RBX1 1503 ±439 - 0.005 ± 0.0004

S0 refers to unmodified substrate, S1 to substrate modified by a single UB, and S2 to substrate modified with two UBs. S0-S1 refers to the transition from unmodified to UB-substrate, and S1-S2 to the transition from UB-substrate to UB-UB-substrate. Values for Km are the best fit values derived from nonlinear regression in Prism, and value for kobs are the best fit values derived from nonlinear regression in Mathematica. The measure of error is the standard error as determined by Prism and Mathematica, respectively, from experiments and curve fits such as those shown in Extended Data Figure 1 (n=2).