Table 1.
Kinetic Constants Measured for Fluoroacetyl-CoA and Acetyl-CoA Hydrolysis by Wild-Type and Mutant FlKsa
| enzyme | fluoroacetyl-CoA
|
acetyl-CoA
|
||||
|---|---|---|---|---|---|---|
| kcat (s−1) | KM (μM) | kcat/KM (M−1 s−1) | kcat (s−1) | KM (μM) | kcat/KM (M−1 s−1) | |
| wild type | (3.9 ± 0.2) × 102 | 8 ± 1 | (5 ± 1) × 107 | (6 ± 1) × 10−2 | (2.1 ± 0.5) × 103 | (3 ± 1) × 101 |
| E50Q | (1.3 ± 0.1) × 10−1 | (1.4 ± 0.1) × 101 | (9 ± 1) × 103 | |||
| H76A | (3.0 ± 0.3) × 10−3 | (2.0 ± 0.7) × 101 | (1.5 ± 0.5) × 102 | |||
| T42A | (4.5 ± 0.9) × 10−1 | (1.1 ± 0.4) × 102 | (4 ± 2) × 103 | |||
| T42S | (1.9 ± 0.4) × 101 | (6 ± 2) × 101 | (3 ± 1) × 105 | (1.0 ± 0.1) × 10−2 | (6.1 ± 0.9) × 101 | (1.6 ± 0.3) × 102 |
| T42C | (1.1 ± 0.2) × 101 | (3.6 ± 1) × 102 | (3 ± 1) × 104 | (1.0 ± 0.1) × 10−2 | (3.9 ± 0.9) × 102 | (1.2 ± 0.6) × 101 |
| F33A | (2.7 ± 0.4) × 101 | (1.5 ± 0.4) × 103 | (1.8 ± 0.5) × 104 | ndb | nd | |
| F36A | (2.7 ± 0.2) × 102 | (7.1 ± 0.8) × 102 | (3.7 ± 0.5) × 105 | (1.0 ± 0.2) × 10−1 | (3 ± 1) × 103 | (2.9 ± 0.9) × 101 |
| V23A | (1.5 ± 0.3) × 102 | (3.0 ± 1.5) × 102 | (5 ± 3) × 105 | (1.0 ± 0.1) × 10−2 | (9 ± 1) × 102 | (1.1 ± 0.2) × 101 |
| L26A | 6.7 ± 0.1 | (1.2 ± 0.2) × 102 | (5.7 ± 0.8) × 104 | nd | nd | |
a
Data are mean ± SE (n=3) as determined from nonlinear curve fitting. Error in the kcat/KM parameter was obtained from propagation of error from the individual kinetic terms.
b
No detectable activity in the presence of up to 10 μM enzyme and up to 2.5 mM acetyl-CoA.