Table II.

Comparison of the P6 and P1′ Specificity of TEV and TVMV Proteases

Enzyme	Substrate	Km (mM)	kcat (s−1)	kcat/Km (mM−1 s−1)	rel. kcat/Km
TVMV protease	TETVRFQ↓SGTRR	0.082 ± 0.020	0.092 ± 0.010	1.12 ± 0.30	1.00
	TETVRFQ↓AGTRR	0.460 ± 0.087	0.112 ± 0.015	0.24 ± 0.06	0.22
	TETVRFQ↓MGTRR	0.671 ± 0.100	0.037 ± 0.003	0.06 ± 0.01	0.05
	TETVRFQ↓YGTRR	0.425 ± 0.087	0.062 ± 0.008	0.15 ± 0.04	0.13
	TETVRFQ↓HGTRR	0.372 ± 0.074	0.012 ± 0.001	0.03 ± 0.01	0.03
	TATVRFQ↓SGTRR	0.244 ± 0.038	0.032 ± 0.002	0.13 ± 0.02	0.12
	TNTVRFQ↓SGTRR	0.142 ± 0.035	0.074 ± 0.008	0.52 ± 0.14	0.46
	TQTVRFQ↓SGTRR	0.169 ± 0.034	0.076 ± 0.008	0.45 ± 0.10	0.40
TEV protease (S219V)	TENLYFQ↓SGTRR	0.093 ± 0.009	0.345 ± 0.009	3.71 ± 0.37	1.00
	TENLYFQ↓AGTRR	0.109 ± 0.011	0.367 ± 0.016	3.36 ± 0.38	0.91
	TENLYFQ↓MGTRR	0.088 ± 0.008	0.198 ± 0.006	2.25 ± 0.22	0.61
	TENLYFQ↓YGTRR	0.060 ± 0.009	0.062 ± 0.004	1.03 ± 0.17	0.28
	TENLYFQ↓HGTRR	0.215 ± 0.056	0.300 ± 0.030	1.40 ± 0.38	0.31
	TANLYFQ↓SGTRR	>1.0	N.D.	0.15 ± 0.01	0.04
	TNNLYFQ↓SGTRR	0.648 ± 0.130	0.244 ± 0.023	0.38 ± 0.08	0.10
	TQNLYFQ↓SGTRR	0.535 ± 0.090	0.109 ± 0.011	0.20 ± 0.04	0.05

ND, not determined.

The relative specificity constants are given as values relative to that obtained with the respective unmodified substrate of the proteases. Substituted residues in the respective TEV and TVMV cleavage sites are in bold. Cleavage site is indicated in arrow.