Table II.

Esterolytic properties of wild-type ETA and mutants

TypeProteinaKm (mM)Vmax (mM min−1)Kcat (s−1)Kcat/Km (s−1mM−1)
ETA7.10 ± 0.890.76 ± 0.0532.8 ± 2.104.64 ± 0.65
V8 protease0.10 ± 0.010.71 ± 0.03376 ± 14.93600 ± 332
Active site mutantsH72AInactiveInactiveInactiveInactive
D120AInactiveInactiveInactiveInactive
S195CInactiveInactiveInactiveInactive
Active site structureP192A6.10 ± 1.40.72 ± 0.0727.0 ± 2.604.50 ± 1.10
maintenance mutantsS211A5.20 ± 1.300.81 ± 0.1035.1 ± 4.106.80 ± 1.90
D-loop mutantsP162ANDb
F163G7.20 ± 1.060.67 ± 0.0528.7 ± 2.204.00 ± 0.67
D164A0.82 ± 0.050.80 ± 0.0234.6 ± 1.0042.0 ± 2.80
D164G2.46 ± 0.170.32 ± 0.017.28 ± 0.232.96 ± 0.22
Substrate specificity mutantsK213AInactiveInactiveInactiveInactive
K213EInactiveInactiveInactiveInactive
K213TInactiveInactiveInactiveInactive
Other mutantsE12ANDb
I62VNDb
R87G6.45 ± 0.260.69 ± 0.0229.9 ± 0.824.64 ± 0.23
  • a All proteins were tested for esterolytic activity with the use of N-t-Boc-l-Glutamic acid α-phenyl ester as a substrate. Esterase activity was measured spectrophotometrically at 270 nm. V8 protease, a known serine protease, served as a positive control.

  • b ND, not yet done.