Steady state kinetic studies for carboxylesterase isoenzymes with CPT-11, NPC, and APC as substrates The substrates, CPT-11 (0–200 μM), NPC (0–200 μM), or APC (0–1000 μM) were incubated in 20 mM Hepes buffer, pH 7.0, with 10% ethylene glycol at 37°C for 2–24 h in 250 μl of reaction volume. The product, SN-38, was extracted with 2 × 5 ml CHCl3 or with Oasis HLB columns and quantitated on an Agilent 1100 HPLC equipped with a fluorescence detector, with excitation set at 375 nm and emission monitored at 560 nm. The concentration of SN-38 was determined from the standard curve generated under identical conditions with each experiment. The substrate consumption was always less than 6%.
Isoenzyme | Substrate | KM a | kcat | Catalytic Efficiency | Normalization of Catalytic Efficiency to CES2 for Each Substrate |
|---|---|---|---|---|---|
| μM | 10-3 min-1 | min-1 mM-1 | |||
| CES1A1 | CPT-11 | 39 ± 3 | 4.9 ± 0.1 | 0.13 | 1 |
| CES2 | CPT-11 | 1.1 ± 0.1 | 13 ± 0.2 | 12 | 100 |
| CES3 | CPT-11 | 137 ± 11 | 0.9 ± 0.04 | 0.0063 | 0.05 |
| CES1A1 | NPC | 80 ± 6 | 1.8 ± 0.06 | 0.023 | 0.5 |
| CES2 | NPC | 3.2 ± 0.2 | 16 ± 0.2 | 5 | 100 |
| CES3 | NPC | 460 ± 50 | 0.26 ± 0.01 | 0.00056 | 0.01 |
|
CES2
|
APC
|
270 ± 50
|
2.45
|
0.015
|
|
↵ a Independent estimations of KM and kcat values were made for each enzyme and substrate. The values for one such experiment with standard error for the fit are reported. The estimations were made by nonlinear regression analysis of the data to the Michaelis-Menten equation (GraFit 4.0; Erithacus Software Ltd.)