Table 3.
Characterization of inactivated sACE-1. Michaelis-Menten kinetic parameters for cleavage of the substrate Mca-RPPGFSAFK(Dnp)-OH by inactivated sACE-1, and percentage of full length sACE-1 remaining, following preincubation of sACE-1 with M-chelate-lisinopril complexes and coreactants
| Complex a | Kinetic parameters for substrate cleavage by inactivated sACE-1 b | % full length sACE-1 remaining after incubation c |
||
|---|---|---|---|---|
| kcat (min−1) | KM (μM) | kcat/KM (min−1μM−1) | ||
| Fe-NTA-lisin | 1,180 ± 80 | 9 ± 2 | 130 ± 30 (48%) | 86 ± 2 |
| Co-NTA-lisin | 1,500 ± 200 | 13 ± 4 | 110 ± 40 (41%) | 91 ± 3 |
| Ni-NTA-lisin | 1,200 ± 100 | 8 ± 2 | 150 ± 40 (56%) | 96 ± 1 |
| Cu-NTA-lisin | 910 ± 10 | 7.5 ± 0.3 | 122 ± 6 (45%) | 90 ± 20 |
| Co-GGH-lisin | 750 ± 20 | 4.6 ± 0.5 | 160 ± 20 (59%) | 80 ± 20 |
| Ni-GGH-lisin | 840 ± 30 | 6.3 ± 0.6 | 130 ± 10 (48%) | 70 ± 20 |
| Cu-GGH-lisin | 150 ± 10 | 7 ± 1 | 22 ± 4 (8.1%) | 50 ± 40 |
| Fe-EDTA-lisin | 1,350 ± 40 | 10.9 ± 0.9 | 120 ± 10 (44%) | 80 ± 30 |
| Co-EDTA-lisin | 780 ± 10 | 11.8 ± 0.3 | 66 ± 2 (24%) | 89 ± 7 |
| Ni-EDTA-lisin | 1,510 ± 50 | 11 ± 1 | 140 ± 10 (52%) | 85 ± 1 |
| Cu-EDTA-lisin | 218 ± 3 | 11.1 ± 0.4 | 19.7 ± 0.9 (7.3%) | 40 ± 30 |
| Fe-DOTA-lisin | 540 ± 20 | 15 ± 1 | 36 ± 4 (13%) | 87 ± 1 |
| Co-DOTA-lisin | 840 ± 10 | 8.7 ± 0.3 | 97 ± 3 (36%) | 91 ± 9 |
| Ni-DOTA-lisin | 610 ± 30 | 5.6 ± 0.7 | 110 ± 20 (41%) | 90 ± 30 |
| Cu-DOTA-lisin | 520 ± 20 | 6.0 ± 0.8 | 90 ± 10 (33%) | 74 ± 1 |
| None | 1,220 ± 50 | 4.5 ± 0.6 | 270 ± 40 (100%) | 100 ± 8 |
a
lisin = lisinopril.
b
kcat values below background, KM values above background, and kcat/KM values below background are shown in bold. The % kcat/KM remaining after each preincubation, relative to control, are shown in parentheses to allow comparison with % full length sACE-1 remaining. The concentration of each complex used for preincubation corresponded to 20% saturation of sACE-1, and ascorbate and H2O2 were each present at 1 mM.
c
Monitored by SDS-PAGE.