Table 2. K_i values for chagasin mutants against T. cruzi cruzipain and human cathepsin L. ND-not determined.

Equilibrium constants for dissociation (K_i) of complexes between cysteine peptidases and chagasin variants. The results are represented as mean values from three independent K_i measurements. The enzymes were assayed for inhibition as described in methods. N.D., not determined

		Ki (pM)
Substitution	Region	cruzipain	cathepsin L
Chagasin	-	7.6 ± 0.67	7.3 ± 1.2
W93A	L6	31 ± 3.5	825 ± 79
Y89S	β7-strand	25 ± 3.1	ND
Y89F	β7-strand	20 ± 0.8	ND
P30A	L2	20 ± 1.7	45 ± 7.3
T31A	L2	317 ± 26	3.9 ± 0.7
T31V	L2	70 ± 4.3	9.7 ± 1.1
T31S	L2	11 ± 0.6	4.9 ± 0.6
T31Y	L2	747 ± 44	6.8 ± 0.9
T32A	L2	16 ± 1.1	14 ± 1.8
T32S	L2	20 ± 1.9	32 ± 2.2
T32E	L2	11 ± 2.0	ND
T32Y	L2	16 ± 0.7	2.7 ± 0.2
T32V	L2	193 ± 13	20 ± 0.8
T31A/T32A	L2	1065 ± 116	1.8 ± 0.4
ΔT31-T32	L2	5868 ± 29	2200 ± 17

The values are mean of three independent K_i determinations ± SE. The conditions of hydrolysis: 50 mM Na₂HP0₄, 100 mM NaCl, 5 mM EDTA, pH 6.5, 5% DMSO, 2.5 mM DTT, at 28°C.