Table 4.

Apparent steady state kinetic constants for perhydrolysis of acetic acid catalyzed by wild-type PFE, L29P PFE and double mutants.^a

Enzyme	Varied substrate	kcat [s-1]	Kmapp [mM]	kcat/Kmapp [s-1 M-1]
Wild-type PFE	Acetic acidb	0.12 ± 0.02	500 ± 100	0.2
	Hydrogen peroxide	0.094 ± 0.002	3.3 ± 0.2	28
L29P PFE	Acetic acid	5.1 ± 0.4	210 ± 60	20
	Hydrogen peroxide	4.4 ± 0.2	1.8 ± 0.2	2000
L29P/F93H PFE	Acetic acid	11 ± 1	610 ± 120	20
	Hydrogen peroxide	9 ± 1	2.7 ± 0.6	3000
L29P/F125A PFE	Acetic acid	10 ± 1	340 ± 80	30
	Hydrogen peroxide	13 ± 1	4.8 ± 0.7	3000
L29P/F57H PFE	Acetic acid	3.3 ± 0.2	380 ± 40	10
	Hydrogen peroxide	6.8 ± 0.2	3.3 ± 0.4	1000

^aKinetic constants were obtained at 23 °C by varying each substrate independently. The fixed acetic acid concentration was 1.4 M except for L29P PFE where it was 500 mM. The fixed hydrogen peroxide concentration was 9.9 mM, which saturates the active site and therefore yields higher values of k_cat.

^bAcetic acid is the likely substrate for the enzyme, so we refer to the substrate as acetic acid even though at pH 5.5 approximately 85 mol% is in the acetate form. The concentrations refer to the sum of both acetic acid and acetate.