TABLE 2.

Enzymatic analysis of JMB19063

The Michaelis-Menten parameters were determined using pNPG as the substrate as described under “Experimental Procedures.” Reactions were incubated for 30 min at enzyme concentrations of 0.5 μg (wild-type) and 5 μg (R587A, F598A, and R587A/F598A) of the recombinant enzyme in a 1-ml reaction volume. Data represent the means ± S.E. of four independent experiments for each enzyme. The R587A/F598A mutant did not have any detectable activity.

Parameter	WT	R587A	F598A
Km (mm)	0.79 ± 0.1	1.7 ± 0.41	3.9 ± 0.19
kcat (s−1)	150 ± 12	56 ± 6.5	1.3 ± 0.28
kcat/Km	198 ± 39	37 ± 15	0.33 ± 0.053