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 2

Kinetic parameters of AprE and AprEΔL₇₅–L₈₂ T₆₆MG₁₀₂D during hydrolysis of s-AAPF-pNa. Reactions were carried out in 100 mM Tris-HCl, pH 8.0, 5 mM CaCl₂, at 37°C, using as substrate s-AAPF-pNa. Values are triplicate determinations in two separate experiments  ± SD.

Enzyme	Kcat (s^-1)	Km (mM)	kcat/Km (s^-1 mM^-1)	% of relative activity
WT AprE	21.8±1.4	1.7±0.2	12.9±0.7	100
AprEΔL75–L82 T66M G102D	2.6±0.4	2.7±0.3	0.96±0.6	7.4