Table 2.

Kinetic and inhibition parameters for SARS PLpro and its mutants on -AMC substrates

SARS PLpro mutant	Kinetic parameter	monoUb-AMC	diUbLys48-AMC (triazole-linked)	diUbLys48-AMC (native)	ISG15-AMC
WT	apparent kcat/KM [M−1s−1]	3.33E+04	1.26E+06	1.01E+06*	5.98E+05
	kcat [s−1]	0.5042 ± 0.02839	42.02 ± 3.872	n/a	9.533 ± 1.218
	KM [μM]	15.12 ± 1.747	33.42 ± 4.869	n/a	15.94 ± 3.172
	fold kcat/KM over monoUb-AMC	1.00	37.70	30.33*	17.93
	Michealis-Menten curve fit (R2)	0.9845	0.9668	n/a	0.9411
F70S E71K H74G (S2 mutant)	kcat/KM [M−1s−1]	-	4.23E+04*	-	2.94E+05
	kcat [s−1]	-	n/a	-	2.748 ± 0.6693
	KM [μM]	-	n/a	-	9.359 ± 3.547
	% kcat/KM of WT (per substrate)	-	3.37	-	49.10
R167S E168R (S1 polar mutant)	kcat/KM [M−1s−1]	-	6.50E+05	-	3.64E+04
	kcat [s−1]	-	65.56 ± 22.38	-	0.318 ± 0.1184
	KM [μM]	-	100.8 ± 42.49	-	8.764 ± 5.614
	% kcat/KM of WT (per substrate)	-	51.63	-	6.08
M209S (S1 hydrophobic mutant)	kcat/KM [M−1s−1]	-	7.06E+05	-	4.20E+05
	kcat [s−1]	-	46.59 ± 10.29	-	4.774 ± 1.263
	KM [μM]	-	66.01 ± 19.61	-	11.88 ± 4.578
	% kcat/KM of WT (per substrate)	-	56.13	-	67.19
WT	Ki [μM] with monoUb**	NI	NI	NI	NI
	Ki [μM] with diUbLys48	2.26 (0.9265)	9.05 (0.8942)	12.07 (0.8892)	3.31 (0.8351)
	Ki [μM] with triUbLys48	-	10.57 (0.9509)	10.11 (0.9066)	4.08 (0.6709)
	Ki [μM] with ISG15	NI	NI	NI	NI

^*Substrate not saturated, k_cat/K_M calculated from slope of linear graph.

n/a – not applicable (k_cat and K_M cannot be independently calculated)

^**K_i values were derived from IC₅₀ values based on the equation K_i = IC₅₀/(S/K_M+1), assuming competitive inhibition, where S is the concentration of the substrate (based on Cer et al. 2009, NAR). Brackets show goodness of fit (R²) of IC₅₀ values obtained from Prism’s log(inhibitor) vs. normalized curve fit. Inhibition curves are shown in Supplemental Figure 6A.

NI – no detectable inhibition (IC₅₀>100μM or data do not converge)