Table S1.

Kinetic values from Ub-AMC cleavage by USP2 variants

USP2	kcat, s−1	KM, M	kcat/KM, M−1s−1	Fold effect*
WT	(1.4 ± 0.1) × 10−1	(1.9 ± 0.3) × 10−6	(8.0 ± 1) × 104	1.0
F462A	(2.9 ± 0.2) × 10−1	(7.0 ± 0.7) × 10−6	(4.1 ± 0.4) × 104	2.0
D466A	(2.0 ± 0.1) ×10−1	(6.0 ± 0.5) × 10−6	(3.3 ± 0.3) ×104	2.4
P474A	(6.0 ± 0.2) × 10−2	(2.5 ± 0.3) × 10−6	(2.4 ± 0.3) × 104	3.3
K503A	(5.7 ± 0.2) × 10−1	(5.1 ± 0.5) × 10−6	(1.2 ± 0.1) × 105	0.7
Y592A	(4.8 ± 0.4) × 10−1	(1.3 ± 0.2) × 10−5	(3.9 ± 0.7) × 104	2.0
D357A†			(5.9 ± 0.2) × 102	135
Q359A†			(3.7 ± 0.1) × 104	2.2
E360A†			(1.3 ± 0.1) × 103	63
L419A†			(7.3 ± 0.2) × 104	1.1
S421A†			(1.2 ± 0.1) × 104	6.9
D436A†			(9.7 ± 0.5) × 103	8.3
L468A†			(3 ± 0.1) × 103	27
K487A†			(2.7 ± 0.1) × 104	3.0
H501A†			(2.9 ± 0.1) × 104	2.8

^*Fold effect was calculated by dividing k_cat/K_M of WT USP2 by the k_cat/K_M of the variant.

^†Only k_cat/K_M could be determined (Materials and Methods).