TABLE 4.
Kinetic parameters for the cleavage of HHL, Z-FHL, and angiotensin I by ACE constructs
Shown is cleavage of HHL, Z-FHL, and angiotensin I by the C-domain, the N-domain, and a number of C-domain mutant constructs. Constants were determined using an ITC assay at 0 and 20 mm NaCl (including 300 mm for the C-domain with HHL) in 50 mm TAPSO (pH 7.5) and 10 μm ZnSO4. Values are shown as the mean of three separate determinations of Km, kcat, and kcat/Km along with S.E.
| Construct | Substrate | [NaCl] | Km | kcat | kcat/Km |
|---|---|---|---|---|---|
| mm | mm | s−1 | mm−1 s−1 | ||
| N-domain | HHL | 0 | 3.910 ± 0.060 | 14.15 ± 0.53 | 3.63 ± 0.14 |
| 20 | 0.720 ± 0.020 | 38.84 ± 0.13 | 53.97 ± 0.15 | ||
| Z-FHL | 0 | 1.360 ± 0.010 | 73.38 ± 0.62 | 54.10 ± 0.47 | |
| 20 | 0.520 ± 0.010 | 509.50 ± 15.61 | 991.25 ± 31.06 | ||
| Angiotensin I | 0 | 0.520 ± 0.010 | 4.04 ± 0.06 | 36.23 ± 0.47 | |
| 20 | 0.077 ± 0.006 | 24.98 ± 1.59 | 325.40 ± 20.54 | ||
| C-domain | HHL | 0 | 1.960 ± 0.020 | 3.26 ± 0.46 | 1.66 ± 0.24 |
| 20 | 1.650 ± 0.030 | 127.00 ± 1.78 | 77.15 ± 1.03 | ||
| 300 | 0.779 ± 0.008 | 606.00 ± 21.86 | 777.50 ± 28.11 | ||
| Z-FHL | 0 | 0.580 ± 0.010 | 80.25 ± 1.20 | 140.00 ± 2.13 | |
| 20 | 0.120 ± 0.010 | 282.00 ± 11.16 | 2372.50 ± 93.67 | ||
| Angiotensin I | 0 | 0.049 ± 0.001 | 1.70 ± 0.11 | 35.28 ± 2.11 | |
| 20 | 0.036 ± 0.001 | 7.38 ± 0.05 | 204.67 ± 1.21 | ||
| R186H | HHL | 0 | 2.740 ± 0.030 | 2.09 ± 0.08 | 0.77 ± 0.03 |
| 20 | 2.060 ± 0.020 | 122.00 ± 1.16 | 59.27 ± 0.62 | ||
| Z-FHL | 0 | 1.000 ± 0.040 | 76.24 ± 2.94 | 76.44 ± 2.94 | |
| 20 | 0.150 ± 0.010 | 467.34 ± 36.45 | 3193.34 ± 247.68 | ||
| Angiotensin I | 0 | 0.085 ± 0.003 | 1.52 ± 0.03 | 17.93 ± 0.29 | |
| 20 | 0.095 ± 0.003 | 7.83 ± 0.19 | 82.65 ± 2.02 | ||
| E403R | HHL | 0 | 4.360 ± 0.050 | 20.48 ± 0.43 | 4.70 ± 0.10 |
| 20 | 1.540 ± 0.040 | 459.00 ± 24.12 | 298.67 ± 15.90 | ||
| Z-FHL | 0 | 0.530 ± 0.010 | 159.34 ± 2.91 | 304.34 ± 5.21 | |
| 20 | 0.540 ± 0.010 | 929.34 ± 14.77 | 1736.67 ± 28.49 | ||
| Angiotensin I | 0 | 0.069 ± 0.003 | 3.96 ± 0.28 | 57.46 ± 4.07 | |
| 20 | 0.061 ± 0.004 | 9.08 ± 0.41 | 123.00 ± 5.42 | ||
| R522Q | HHL | 0 | 2.820 ± 0.020 | 6.18 ± 0.17 | 2.20 ± 0.06 |
| 20 | 1.680 ± 0.010 | 4.80 ± 0.18 | 2.87 ± 0.11 | ||
| Z-FHL | 0 | 0.110 ± 0.010 | 92.24 ± 2.16 | 860.34 ± 20.01 | |
| 20 | 0.200 ± 0.010 | 124.67 ± 9.36 | 653.34 ± 47.73 | ||
| Angiotensin I | 0 | 0.092 ± 0.003 | 12.98 ± 0.29 | 141.75 ± 3.20 | |
| 20 | 0.077 ± 0.003 | 13.75 ± 0.84 | 179.50 ± 11.14 | ||
| R522K | HHL | 0 | 3.720 ± 0.020 | 5.31 ± 0.19 | 1.43 ± 0.06 |
| 20 | 2.250 ± 0.020 | 2.03 ± 0.05 | 0.91 ± 0.02 | ||
| Z-FHL | 0 | 0.570 ± 0.010 | 126.00 ± 1.00 | 223.25 ± 1.94 | |
| 20 | 0.880 ± 0.020 | 193.50 ± 5.13 | 220.75 ± 5.97 | ||
| Angiotensin I | 0 | 0.130 ± 0.002 | 0.83 ± 0.03 | 6.37 ± 0.21 | |
| 20 | 0.119 ± 0.002 | 3.79 ± 0.07 | 32.05 ± 0.53 |