Engineering and Directed Evolution of a Ca²⁺ Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu₇₅–Leu₈₂ to Enzyme Activity

Table 3

Thermal inactivation parameters (k _i and t _1/2 *) of wild-type AprE and AprEΔL₇₅–L₈₂ T₆₆M G₁₀₂D (Var1).

Assay condition	WT 𝑘𝑖 (10^-4 min^-1)	WT 𝑡1/2 (min)	Var1 𝑘𝑖 (10^-4 min^-1)	Var1 𝑡1/2 (min)
5 mM Ca^2+, 50°C	8±2	856	51±4	135
0.1 mM Ca^2+, 100 mM NaCl, 50°C	130±16	52	2220±141	3.1
0.1 mM EDTA, 100 mM NaCl, 50°C	2700±170	2.6	3255±32	2.1
5 mM Ca^2+, 65°C	490±100	14	4550±9	1.5

* 𝑡_1/2 were obtained considering an 𝐴_𝑅=50% and using (3) (Materials and Methods). Enzymes were dissolved in 100 mM Tris-HCl, pH 8.0. Values are triplicate determinations in two separate experiments  ± SD.