TABLE 2.

Kinetic parameters and thermal stability of the Pce variant proteins^a

Protein	Tm (°C)	Km (mM)	kcat (s−1)
Wild type	46/64	0.631	5.11
K217E	44/64	0.608	6.38
K245E	44/67	0.106	1.28
K259E	44/65	0.795	6.38
K263E	42/66	0.470	6.38
K267E	43/65	0.608	6.38
K284E	44/63	0.278	1.91
K319E	43/64	0.743	5.11
K259E/K319E	43/62	0.222	3.83
K267E/K319E	40/64	0.743	6.38
K259E/K267E	38/62	0.8	11.49
K259E/K267E/K319E	46/64	0.608	8.94

^aT_m values were determined from a protein thermal stability assay using 3.5 μg of protein and 5 mM TCEP, with SYPRO orange as the environmentally reporter dye. PCho esterase activity measurements were performed using PBS (pH 7.4) at 37°C, and data were fitted using the Michaelis-Menten equation and the Lineweaver Burk plot from which K_m and k_cat values were determined.