TABLE 1.
Steady-state kinetic analysis of wild-type and mutant T. brucei AdoMetDC enzymes
kcat/Km (m−1 s−1) values were determined through linear regression (slope) of the velocity versus substrate plots, and the highest substrate concentration tested was used to determine a lower estimate of Km and kcat. The error represents the S.D. for n = 3 replicates.
| Enzyme | 5 mm putrescine |
No putrescine |
||||
|---|---|---|---|---|---|---|
| kcat | Km | kcat/Km | kcat | Km | kcat/Km | |
| s−1 | mm | m−1 s−1 | s−1 | mm | m−1 s−1 | |
| WT AdoMetDC homodimer | 0.0065 ± 0.0004 | 0.14 ± 0.04 | 46 ± 12 | 0.0058 ± 0.0003 | 0.40 ± 0.06 | 15 ± 2 |
| WT AdoMetDC·prozyme | 0.21 ± 0.01 | 0.13 ± 0.02 | (1.7 ± 0.3) × 103 | 0.25 ± 0.02 | 0.14 ± 0.02 | (1.8 ± 0.5) × 103 |
| Δ16 AdoMetDC homodimer | >0.03 | >2 | 16 ± 3a | >0.032 | >2 | 16 ± 11a |
| Δ16 AdoMetDC·prozyme | 0.017 ± 0.001 | 0.60 ± 0.1 | 28 ± 5 | 0.014 ± 0.001 | 0.80 ± 0.1 | 18 ± 3 |
| AdoMetDC(KDS-AAA)·prozyme | 0.33 ± 0.07 | 0.60 ± 0.3 | (5.7 ± 0.3) × 102 | 0.071 ± 0.005 | 0.37 ± 0.07 | (1.9 ± 0.4) × 102 |
| AdoMetDC(LSL-AAA)·prozyme | 0.011 ± 0.001 | 1.2 ± 0.2 | 9.0 ± 1.0 | >0.01 | >2 | 5.1 ± 0.5a |
| AdoMetDC(MAM-AAA)·prozyme | 0.026 ± 0.002 | 0.31 ± 0.08 | 85 ± 22 | >0.01 | >2 | 4.8 ± 0.1a |
| AdoMetDC(WGS-AAA)·prozyme | 0.10 ± 0.01 | 0.30 ± 0.1 | (2.8 ± 0.1) × 102 | 0.18 ± 0.04 | 1.0 ± 0.5 | (1.7 ± 0.8) × 102 |
| AdoMetDC(L8A)·prozyme | 0.12 ± 0.003 | 0.45 ± 0.03 | (2.5 ± 0.2) × 102 | >0.03 | >2 | 16.6 ± 0.5a |
| AdoMetDC(S9A)·prozyme | 0.29 ± 0.02 | 0.90 ± 0.1 | (3.4 ± 0.5) × 102 | 0.23 ± 0.01 | 1.4 ± 0.1 | (1.8 ± 0.2) × 102 |
| AdoMetDC(L10A)·prozyme | 0.084 ± 0.009 | 0.22 ± 0.08 | (3.8 ± 0.1) × 102 | >0.10 | >2 | 50 ± 9a |
| WT AdoMetDC·Δ25 prozyme | 0.15 ± 0.01 | 0.20 ± 0.04 | (7.7 ± 0.2) × 102 | 0.23 ± 0.04 | 0.60 ± 0.20 | (4 ± 1) × 102 |
| Δ16 AdoMetDC·Δ25 prozyme | >0.004 | >2 | 2.2 ± 0.1a | >0.006 | >2 | 3.1 ± 0.3a |
a Kinetic experiments in which substrate saturation was not observed.